Neuregulin for the treatment and/or prevention of tumors of the nervous system

ABSTRACT

A polypeptide is provided that includes an EGF-like domain of a neuregulin protein. A nucleic acid encoding for the polypeptide is also provided. A gene therapy vector inclusive of the nucleic acid and genetically modified cells expressing said polypeptide are also provided. The medical use of the polypeptide, the nucleic acid, the gene therapy vector or the cell for the treatment of tumours of the nervous system are also provided. In particular the treatment of tumours of the cranial or peripheral nerves, tumours associated with neurofibromatosis, schwannomas, neurofibromas and malignant nerve sheath tumours are provided.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/999,851, filed 20 Aug. 2018; that in turn is a US National Phaseof PCT Application Serial Number PCT/EP2017/059245, filed 19 Apr. 2017;that in turn claims priority of European Application Serial NumberEP16165991.7 filed 19 Apr. 2016, the contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The invention relates to a polypeptide, wherein said polypeptidecomprises or consists of an EGF-like domain of a neuregulin protein. Theinvention relates to a nucleic acid encoding for said polypeptide, agene therapy vector comprising said nucleic acid and geneticallymodified cells expressing said polypeptide. The invention relates to themedical use of said polypeptide, said nucleic acid, said gene therapyvector and/or said cell for the treatment of tumours of the nervoussystem, in particular for the treatment of tumours of the cranial orperipheral nerves, tumours associated with neurofibromatosis,schwannomas, neurofibromas and malignant nerve sheath tumours.

BACKGROUND OF THE INVENTION

Neurofibromatosis (NF) is a genetic disorder, which results in a medicalcondition affecting the nervous system, skin, bones, and muscles andmanifests itself often by the presence of multiple soft nodules,neurofibromas and is associated with hyperpigmented spots. The diseaseis inherited as an autosomal dominant trait. Neurofibromatosis exists inthree different forms: Neurofibromatosis type 1 (NF1), known as VonRecklinghausen's disease, Neurofibromatosis type 2 (NF2), andNeurofibromatosis type 3 (NF3), often referred to schwannomatosis. Thedisorders occur as a result of genetic defects. The gene for NF1, hasbeen located on chromosome 17 and for NF2, on chromosome 22. A candidategene for schwannomatosis (NF3) is SMARCB1, which is located onchromosome 22 in proximity to the gene for NF2.

NF1, also known as von Recklinghausen Disease, is a hereditary diseasewith a birth incidence of one in 2500-3000. It associated a mutation ofthe gene Nf1 which is located on human chromosome 17q11.2 andcompromises 60 exons spanning 350 kb of genomic DNA. The Nf1 geneencodes for neurofibromin 1, a tumour suppressor postulated to functionin part as a Ras GTPase-activating protein and negatively regulating theRas oncogene signal transduction pathway. Neurofibromin is expressedthroughout the nervous system and negatively influences cellproliferation.

NF1 is characterized by a variety of disorders including the appearanceof café-au-lait patches (skin discolorations), skin-fold freckling,Lisch nodules, plexiform neurofibromas, cutaneous neurofibromas,scoliosis, pseudo arthrosis of the tibia or renal artery stenosis. It isfurther associated with skeletal dysplasia, vascular dysplasia, learningdisabilities, epilepsy, optic pathway glioma, cerebral glioma, seizuresand other tumours of the neural crest origin, such as pheochromocytomas.NF1 may also result in malignant peripheral nerve sheath tumour (MPNST).

The occurrence of neurofibromas in NF1 especially on the spinal cord mayresult in secondary abnormalities of the CNS due to the exertedpressure. Cognitive impairment, multiple sclerosis and epilepsy havebeen associated with NF1. The most common neurological symptom in NF1for children is a cognitive disability that is characterized by belowaverage intelligent quotient. In NF1, neurofibromas most commonly growon the skin or on the nerve to the eye. A tumour that grows on the nerveto the eye is commonly called an optic glioma, and if it grows largeenough can cause problems with vision, including blindness. Opticpathway gliomas are frequent in younger children, but may also appear inolder patients. If untreated it may lead to visual loss and or visualdefects such as strabismus, proptosis, afferent pupillary defect opticdisc oedema or atrophy. Chemotherapy and radiotherapy present potentialtreatments. They exhibit however side effects. Lisch nodules in NF1 mayfurther affect the iris or the eyelid. Idiopathic congenital ptosis andcongenital acquired glaucoma also occur in NFL

The term neurofibroma has been coined by von Recklinghausen and refersto benign tumours. These tumours can grow anywhere in the body wherethere are nerve cells. This includes nerves just under the surface ofthe skin, as well as nerves deeper within the body, spinal cord and/orbrain. Neurofibromas usually manifest in benign, focal cutaneous orsubcutaneous lesions or plexiform tumours. Often neurofibromasoriginated from neoplastic Schwann cells, fibroblast and perineuralcells, which are embedded in a collagen fibre matrix. Neurofibroma canrarely be surgically removed from the nerves without damaging thenerves. Most commonly neurofibroma occur on the skin and are highlyvisible. Patients suffering from neurofibroma therefore have a tendencyfor social isolation and may necessitate psychological care.

Moreover subjects afflicted with NF1 have a high risk of developingmalignant peripheral nerve sheath tumours (MPNST). MPNST develop oftenfrom prior plexiform or focal subcutaneous neurofibroma. Due to a highprobability of metastasis MPNST are particularly aggressive and havepoor prospects.

NF2 is an autosomal-dominant multiple neoplasia syndrome that resultsfrom mutations in the Nf2 tumour suppressor gene. The Nf2 tumoursuppressor gene is located on human chromosome 22 and comprises 17exons. The Nf2 gene encodes for a 69 kDa product referred to asmoesin-ezrin-radixin-like protein (merlin), also referred toneurofibromin 2 or schwannomin. Merlin is a membrane-cytoskeletonscaffolding protein that links the membrane or membrane glycoproteins tothe actin cortex of the cells. Human merlin, which exists in twoisoforms, is expressed throughout the nervous tissues and localizes atcell-cell adherens junctions. Its function as a tumour suppressor isthought to evoke from a contact-mediated inhibition of cellproliferation.

NF2, is characterized by bilateral vestibular schwannomas withassociated symptoms of tinnitus, hearing loss and balance dysfunction.Other findings include schwannomas of other cranial and peripheralnerves, meningiomas and juvenile posterior subcapsular cataract.

NF3, also referred to as Schwannomatosis is a more recently describedtype of neurofibromatosis characterized by multiple cutaneousschwannomas, central nervous system tumours, and other neurologicalcomplications.

Schwann cell tumours, referred to as schwannomas, are nerve sheathneoplasms that appear sporadically and in association with geneticsyndromes such as Schwannomatosis or Neurofibromatosis type 2 (NF2).Despite their predominantly benign nature, these tumours often have adevastating impact on patients' life quality, as treatment options areoften limited to tumour resection by surgery—therein endangeringlong-term nerve functionality. In the hereditary tumour syndrome NF2with an incidence of 1 in 33,000 live births (Evans et al., 2010), thisoften means lifelong deafness as schwannomas predominantly appear at thevestibulocochlear nerve (vestibular schwannoma). Bilateral vestibularschwannomas occur particularly often in NF2 with a frequency of 90-95%(Asthagiri et al., 2009). Moreover meningiomas are the second mostcommon tumours associated with NF2, wherein intracranial meningiomas andintradural extramedullary spinal meningiomas are particularly frequent.Furthermore spinal cord ependymomas are associated with NF2. Mostsubjects afflicted with NF2 will moreover develop peripheral neuropathy,which often result from tumours comprising the function of the nervoussystem. Skin tumours including skin plaques, subcutaneous tumours andintradermal tumours are also associated with NF2.

The multifocality of tumours appearing during the lifetime ofindividuals with Nf2 gene mutations further indicates the urgent needfor pharmacological therapies that enable systemic tumour control inparticular in NF2 patients (Asthagiri et al., 2009).

Like for other orphan diseases, existing oncology drugs were repurposedfor NF2 disease in several clinical trials over the past years (Bakkeret al., 2016). So far, only the VEGF-inhibitory antibody bevacizumabshowed limited efficacy for schwannoma growth control and hearingimprovement (Plotkin et al., 2009); but it is accompanied by severe sideeffects in long-term use (Mautner et al., 2010; Slusarz et al., 2014).However, disease-targeted therapies addressing NF2-related biologicalspecifics are currently still not available.

Neuregulin 1 (NRG1) is a trophic factor containing an epidermal growthfactor (EGF)-like domain that signals by stimulating ErbB receptortyrosine kinases (Mei and Xiong, 2008). The biological effects of NRG1are highly versatile but its role in the peripheral nervous system (PNS)has been extensively studied in the past years. During PNS development,axonal NRG1 regulates myelin thickness by determining Schwann cell fate(Michailov et al., 2004). While the growth factor-like molecule NRG1 isdispensable for maintenance of myelin sheaths after development, itgains importance again for nerve repair processes and especiallyre-myelination after injury (Fricker et al., 2011). Importantly, arecent publication demonstrated that myelination is not only controlledby membrane-retained NRG1 type III (Jessen and Mirsky, 2005), but alsoin a paracrine manner via proteolytic liberation of the EGF-like domain(Fleck et al., 2013).

Consistently, the soluble EGF-like domain of NRG1 could rescue ahypomyelination zebrafish mutant (Fleck et al., 2013) and was capable ofameliorating the phenotype of a Charcot-Marie-Tooth 1A animal model(Fledrich et al., 2014). Previously it was postulated that not onlySchwann cell-intrinsic signalling cues but also extracellular factorsmay play a role in schwannomas (Schulz et al., 2014a). Furthermore, itwas demonstrated that the axon surface molecule Neuregulin1 type III(NRG1 type III) shows reduced expression in nerves with a conditionalnf2 gene knockout (Schulz et al., 2014b).

Stove et al. discusses implications of neuregulins as EGF-like ligandsin the activation of human epidermal growth factor receptors (HERs) andtheir influence on cancer progression. WO 01/89568 A proposes the useneuregulin-2-polypeptides for treating multiple sclerosis, spinalmuscular atrophy, nerve injury or Alzheimer's disease, by increasingmitogenesis, survival, growth or differentiation of cells expressing anerbB receptor that is selective for neuregulin-2. WO 01/89568 A alsoproposes a treatment of a glial tumor by inhibiting the proliferation ofthe tumor cells. The treatment involves the administration of anantibody that inhibits the binding of an NRG-2 polypeptide to a receptorpresent on the surface of the tumor cell.

As described herein, Neuregulin administration represents an effectivetherapeutic approach for treating tumours of the nervous system andassociated medical conditions, in particular in relation toneurofibromatosis. The therapeutic administration of Neuregulin for thetreatment of these medical conditions has not been previously described.

SUMMARY OF THE INVENTION

In light of the prior art the technical problem underlying the presentinvention is to provide alternative and/or improved means for thetreatment of tumours of the nervous system, such as medical conditionsassociated with neurofibromatosis, tumours of the cranial or peripheralnerves, schwannomas, vestibular schwannomas, meningioma or malignantnerve sheath tumours.

This problem is solved by the features of the independent claims.Preferred embodiments of the present invention are provided by thedependent claims.

The invention therefore relates to a polypeptide for use as a medicamentin the treatment and/or prevention of a tumour of the nervous system,wherein said polypeptide comprises or consists of an EGF-like domain ofa neuregulin-1 protein. The invention also relates to a method for thetreatment of a subject afflicted by a tumour of the nervous system orfor the prevention and/or reduction of the risk of development of atumour of the nervous system in a subject at risk thereof, wherein themethod comprises the administration of a polypeptide comprising orconsisting of an EGF-like domain of a neuregulin-1 protein to saidsubject.

Neuregulin proteins (NRG) belong to the family of epidermal growthfactors (EGF) and comprise an EGF-like domain. In mammals the genesneuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3) andneuregulin 4 (NRG4) encode for neuregulin proteins and comprise EGF-likedomains that may be used in embodiments of the present invention.

According to a preferred embodiment of the present invention, the termneuregulin protein refers to the proteins encoded by the gene NRG1 (GeneID 3084, http://www.ncbi.nlm.nih.gov/gene/3084). Through alternativesplicing, the NRG1 gene encodes for different isoforms. In furtherembodiments the term neuregulin protein refers to all isoforms encodedby NRG1 as published on http://www.ncbi.nlm.nih.gov/gene/3084.

In preferred embodiments the term neuregulin protein therefore refers tothe following isoforms encoded by NRG1: human pro-neuregulin-1,membrane-bound isoform HRG-beta1c (NCBI references NM_001159995.1 andNP_001153467.1), human pro-neuregulin-1, membrane-bound isoform ndf43c(NCBI references NM_001159996.1 and NP_001153468.1), humanpro-neuregulin-1, membrane-bound isoform HRG-beta1b (NCBI referencesNM_001159999.1 and

NP_001153471.1), human pro-neuregulin-1, membrane-bound isoformHRG-beta1d (NCBI references NM_001160001.1 and NP_001153473.1), humanpro-neuregulin-1, membrane-bound isoform HRG-gamma2 (NCBI referencesNM_001160002.1 and NP_001153474.1), human pro-neuregulin-1,membrane-bound isoform ndf43b (NCBI references NM_001160004.1 andNP_001153476.1), human pro-neuregulin-1, membrane-bound isoformHRG-beta3b (NCBI references NM_001160005.1 and NP_001153477.1), humanpro-neuregulin-1, membrane-bound isoform HRG-gamma3 (NCBI referencesNM_001160007.1 and NP_001153479.1), human pro-neuregulin-1,membrane-bound isoform HRG-beta2b (NCBI references NM_001160008.1 andNP_001153480.1), human pro-neuregulin-1, membrane-bound isoformHRG-gamma (NCBI references NM_004495.3 and NP_004486.2), humanpro-neuregulin-1, membrane-bound isoform HRG-beta1 (NCBI referencesNM_013956.3 and NP_039250.2), human pro-neuregulin-1, membrane-boundisoform HRG-beta2 (NCBI references NM_013957.3 and NP_039251.2), humanpro-neuregulin-1, membrane-bound isoform HRG-beta3 (NCBI referencesNM_013958.3 and NP_039252.2), human pro-neuregulin-1, membrane-boundisoform SMDF (NCBI references NM_013959.3 and NP_039253.1), humanpro-neuregulin-1, membrane-bound isoform ndf43 (NCBI referencesNM_013960.3 and NP_039254.1), human pro-neuregulin-1, membrane-boundisoform GGF2 precursor (NCBI references NM_013962.2 and NP_039256.2) andhuman pro-neuregulin-1, membrane-bound isoform HRG-alpha (NCBIreferences NM_013964.3 and NP_039258.1). The use of sequence variants ofneuregulin proteins that exhibit functional analogy to the unmodifiedhuman isoform is also encompassed by the present invention.

The EGF-like domain of neuregulin-1 exhibits a high therapeuticefficiency for the treatment of tumours of the nervous system. It isknown that the EGF-like domain of neuregulin-1 takes part in signaltransduction by stimulating the ErbB receptor tyrosine kinase ErbB2. Itis particularly preferred for the invention that the polypeptidecomprising the EGF-like domain is soluble. The soluble EGF-like domainof NRG1 has previously been reported to be effective in the treatment ofcardiovascular diseases (Mendes et al. 2013). However its potency in thetreatment of tumours or cancer in particular of tumours of the nervoussystem has not been suggested nor described.

It was therefore surprising that the EGF-like domain of NRG1 doeseffectively inhibit the growth of tumours of the nervous system. Asunraveled by the inventors and demonstrated in the examples herein, theEGF-like domain of neuregulin-1 acts preferably on the tumorous cellsthrough a differentiation inducing signal. The differentiation stage oftumours and in particular of tumours of the nervous system is stronglyassociated with its behaviour. In general, an immature, lessdifferentiated tumour is more aggressive than the more differentiatedcounterpart. Tumours that are differentiated resemble more closely theoriginal healthy tissue they originate from. A tumour that resembles theoriginal tissue to a lesser extent is termed poorly differentiated, oranaplastic. In general, and in particular for tumours of the nervoussystem, poorly differentiated tumour cells exhibit a higher motility andhigh proliferation rates. Due this properties de-differentiated tumourscells outcompete and invade the healthy surrounding tissues. Anachievement of the invention is that it was recognized that the EGF-likedomain of NRG1 may act as a differentiation inducer on tumour cells ofthe nervous system. By differentiation of the tumour cells theproliferation rate can be reduced and tumour growth efficiently stoppedor even reversed.

In particular, the polypeptide comprising the EGF-like domain ofneuregulin enhances the susceptibility of tumour cells of the nervoussystem to mitogenic signals impeding cell proliferation. Treatment oftumour cells with the polypeptide comprising the EGF-like domain ofneuregulin results in reduced rates of cell division and tumourexpansion. In particular for Schwann cells, polypeptides comprising theEGF-like domain of an axon surface protein NRG1, according to preferredembodiments of the invention, are particularly efficient indifferentiating Schwann cells. By differentiation of the tumorousSchwann cells physiological myelinating of the nerves can be restoredand abnormal growth resulting in Schwannomas can be prevented, stoppedor reversed.

The differentiation of the tumour cells by the EGF-like domain of NRG1moreover reduces the mobility of the tumour cells. For instancemalignant schwannomas often arise spontaneously in adult patients or mayoccur with increased frequency in patients with Neurofibromatosis typeI. Often these tumours exhibit a low degree of differentiation, visibleby a loose tissue connection, and augmented frequency of metastasis. Bytreating benign neurofibromas with the polypeptide according to theinvention the tumour can be contained and the development of malignantperipheral nerve sheath tumor (MPNST) as well as subsequent metastasisprevented.

Moreover, in some embodiments of the invention the polypeptide describedherein is used in a preventive therapy of subjects afflicted by a riskof developing a tumour of the nervous system. Without being limited bytheory, the polypeptide advantageously impedes the de-differentiationprocess believed to be at the origin of tumour formation. Through thisapproach newly developing tumours with at an early stage that do not yetresult in symptoms of a disease may be efficiently prevented fromdeveloping into severe tumours. Due to the low side effects intreatments using the polypeptide according to the invention, long termtherapies are possible. For a preventive approach it may even bepreferred to administer the polypeptide at a regular interval overmultiple weeks, months, or years without significant detrimental sideeffects.

In a preferred embodiment of the invention the polypeptide for use as amedicament is characterized in that the polypeptide is a solublefragment of a neuregulin protein. A preferred embodiment of theinvention therefore refers to a soluble polypeptide comprising theEGF-like domain of neuregulin. According to the invention “soluble”preferably refers to fragments of proteins or polypeptides that are notpermanently membrane bound. Many isoforms of neuregulin proteins aretransmembrane proteins, which are thus membrane-retained. The preferredembodiment of a soluble fragment of a neuregulin protein thereforerefers preferably to fragments of a transmembrane neuregulin, which arenot membrane-retained.

It is particularly preferred that the soluble fragment of a neuregulinprotein refers to the extracellular domain of a neuregulin protein thatcomprises the EGF-like domain. The soluble EGF-like domain of neuregulinis particularly potent for the treatment of tumours of the nervoussystem. The preferred embodiment allows for an efficient distribution ofthe therapeutic polypeptide throughout the organism of a treatedsubject. In particular by systemic administration e.g. by intravenousinjection the soluble polypeptide can be transported by the vascularsystem throughout the body of a treated subject. Moreover, proteinclustering or precipitation can be avoided. It was particularlysurprising that soluble fragments of the neuregulin protein exhibit ahigh tendency to localize to tumours of the nervous system. Solublepolypeptides comprising the EGF-like domain of a neuregulin protein showtherefore an augmented specificity and reduced side effects.Advantageously the concentration of the polypeptide comprising theEGF-like domain may potentially be increased to high therapeutic levelswithout introducing unwanted side effects.

A particular preferred embodiment of a soluble fragment of a neuregulinprotein is a polypeptide according to SEQ ID NO 9 as well as functionalanalogous polypeptides thereof. In a preferred embodiment thepolypeptide has an amino acid sequence identity of at least 80%,preferably of at least 90%, to SEQ ID NO 9. These polypeptides arepreferably characterised by a high stability and biocompatibility.Additionally, since the degradation rate of these peptides is expectedto be relatively low, they are expected to show a prolongedtherapeutically effect.

In a preferred embodiment the polypeptide for use as a medicamentaccording to the invention is characterized in that neuregulin proteinis human neuregulin 1 type I, human neuregulin 1 type II and/or humanneuregulin 1 type III, or a fragment thereof.

In a preferred embodiment of the invention the polypeptide for use as amedicament is characterized in that the polypeptide comprises orconsists of an amino acid sequence according to SEQ ID NO 9-14 or anamino acid sequence with an identity of at least 80%, preferably of atleast 90%, to any one of SEQ ID NO 9-14.

In a preferred embodiment of the invention the polypeptide for use as amedicament is characterized in that the polypeptide consists of an aminoacid sequence according to SEQ ID NO 9 or of an amino acid sequence withan identity of at least 80%, preferably of at least 90% to SEQ ID NO 9.

The polypeptide as described herein is intended for use as a medicamentin the treatment of a tumour of the nervous system, wherein the tumouris preferably a tumour of the cranial or peripheral nerves. Preferably atumour of the cranial or peripheral nerves refers to a schwannoma, aneurofibroma, a perineurioma or a malignant nerve sheath tumour.

In one embodiment the polypeptide for use as a medicament as describedherein is characterised in that the tumour to be treated is a malignantnerve sheath tumour.

In one embodiment the polypeptide for use as a medicament as describedherein is characterised in that the tumour to be treated is aschwannoma.

In one embodiment the polypeptide for use as a medicament as describedherein is characterised in that the tumour to be treated is aneurofibroma.

It is to be understood the preferred embodiments of the polypeptide foruse as a medicament as described herein are preferably administered inthe method for treating a subject or preventing/reducing risk a subjectafflicted by or in risk of the development of a tumour of the nervoussystem.

The invention therefore encompasses a method for treating and/orpreventing, or reducing the risk of development of, one or more of thetumours listed herein. In some embodiments the present invention relatesto an NRG1-replacement therapy.

In one embodiment the polypeptide for use as a medicament as describedherein is characterised in that treatment and/or prevention of medicalconditions associated with genetic deficiency in Neurofibromatosis type1, Neurofibromatosis type 2 and/or Neurofibromatosis type 3 is intended.

Specific examples of medical conditions that are associated withneurofibromatosis, which are preferably treated by the use of thepolypeptide, the nucleic acid and/or the gene therapy vector accordingto the invention or preferred embodiments thereof include but are notlimited to neurofibromas, plexiform neurofibromas, cutaneousneurofibroma, epilepsy, learning disabilities, optic pathway glioma aswell as visual losses associated with these, cerebral glioma, seizuresand other tumours of the neural crest origin, such as pheochromocytomas,malignant peripheral nerve sheath tumours, bilateral vestibularschwannomas with associated symptoms of tinnitus, hearing loss andbalance dysfunction, schwannomas of other cranial and peripheral nerves,meningiomas, intracranial meningiomas, intradural extramedullary spinalmeningiomas, skin tumours including skin plaques, subcutaneous tumoursand intradermal tumours, ependymomas or ependymomas of the spinal cord.

It was surprising that polypeptides according to the invention andpreferred embodiments thereof are efficient for treating these medicalconditions associated with neurofibromatosis. The approach to administera polypeptide comprising an EGF-like domain of a neuregulin proteinaccording to the invention constitutes a departure of the state of theart. For example, a person skilled in the art may have attempted totreat NF1, NF2 and/or NF3 by replacing the proteins encoded by NF1 gene,NF2 gene and/or NF3 genes. For instance, a person skilled in the art mayhave attempted to provide exogenous merlin inside of cells of a subjectafflicted by NF2. However the use of the EGF-like domain of an axonsurface protein like NRG1 for the treatment of diseases associated withneurofibromatosis is an alternative and novel strategy. The strategy issurprisingly effective. The provision of an extracellular solublepolypeptide has therefore therapeutic advantages over attempts toreplace the NF gene encoded proteins inside of the cells as e.g. merlininside of Schwann cells in NF2 patients. In particular thetherapeutically effective dosage of the polypeptides can be more easilyand securely controlled and side effects can be minimized.

In a further preferred embodiment the invention relates to a nucleicacid molecule for use as a medicament in the treatment and/or preventionof a tumour of the nervous system, wherein the nucleic acid moleculeencodes a polypeptide according to the invention. The inventiontherefore also relates in a further preferred embodiment to a method fortreating a subject afflicted by or in risk of the development of atumour of the nervous system, wherein the method comprises theadministration of a nucleic acid molecule encoding a polypeptideaccording to the invention or preferred embodiments thereof.

In a preferred embodiment the nucleic acid molecule is used for thetreatment and/or prevention a tumour of the cranial or peripheralnerves. In a preferred embodiment the nucleic acid molecule is used forthe treatment and/or prevention a malignant nerve sheath tumour. In apreferred embodiment the nucleic acid molecule is used for the treatmentand/or prevention a schwannoma. In a preferred embodiment the nucleicacid molecule is used for the treatment and/or prevention aneurofibroma. In a preferred embodiment the nucleic acid molecule isused for the treatment and/or prevention of tumours associated withgenetic deficiency in neurofibromatosis type 1, neurofibromatosis type 2and/or neurofibromatosis type 3.

In a further preferred embodiment the invention relates to a genetherapy vector for use as a medicament in the treatment and/orprevention of a tumour of a nervous system comprising said nucleic acidmolecule or preferred embodiments thereof. In this further preferredembodiment the invention thus also relates to a method for treating asubject afflicted by or in risk of the development of a tumour of thenervous system, wherein the method comprises the administration of genetherapy vector comprising said nucleic acid molecule or preferredembodiments thereof.

In a preferred embodiment the gene therapy vector is used for thetreatment and/or prevention tumour of the cranial or peripheral nerves,a malignant nerve sheath tumour, a schwannoma and/or a neurofibroma.

In particularly preferred embodiments the gene therapy vector is usedfor the treatment and/or prevention of tumours associated with geneticdeficiency in neurofibromatosis type 1, neurofibromatosis type 2 and/orneurofibromatosis type 3.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are presented in order to describe particularembodiments of the invention, by demonstrating a practicalimplementation of the invention, without being limiting to the scope ofthe invention or the concepts described herein.

FIGS. 1A-C: Schematic representations of an experimental protocol toassess the efficacy of rhNRG1 treatment and experimental results forserum levels of rhNRG1 and body weight of treated animals based on adefined study protocol (FIG. 1A), a plot of rhNRG1 levels afterinjection (FIG. 1B), with body weight plotted as a function of time(FIG. 1C);

FIGS. 2A-D: Experimental results for functional nerve regeneration afternerve crush by treatment of rhNRG1 for foot-based angle plotted as afunction of time (FIG. 2A), Nf2flox;PO-Cre;Nefh-Cre mutant micereceiving continuous intraperitoneal rhNRG1 injections every other daydisplayed significantly improved recovery compared to mice withouttreatment or saline control as shown as a plot of foot-based angleplotted as a function of time (FIG. 2B), the local administration ofrhNRG1 using absorbable collagen sponges was able to increase functionalregeneration in Nf2flox;PO-Cre;Nefh-Cre mutant mice compared to controltreatment as shown as a plot of foot-based angle plotted as a functionof time (FIG. 2C), absorbable collagen sponges (ACS) cubes incubatedwith solutions containing different concentrations of rhNRG1 are plottedas a function of time (FIG. 2D);

FIGS. 3A-B: Experimental results for sciatic nerve diameter followingrhNRG1 treatment, three months after crush injury wildtype animals nervediameter is plotted for vehicles alone and with administration of rhNRG1(FIG. 3A), minor effects were seen in Nf2flox;PO-Cre;Nefh-Cre mutantmice receiving a local therapy of rhNRG1 using absorbable collagensponges (rhNRG1 ACS) when compared to Nf2flox;PO-Cre;Nefh-Cre mutantmice receiving the control therapy based on nerve diameter (FIG. 3B);

FIGS. 4A-C: Experimental results for signalling proteins with sciaticnerve lysates FIG. 4A, are micrographs of sciatic nerve cross sectionsof indicated cohorts 3 months after crush injury for listing statins ormarkers, the scale bar represents 20 μm, FIG. 4B is an immunoblot ofsciatic nerve lysates for Erk1/2, phospho-Erk1/2 (pErk1/2), myelinprotein zero (MPZ), myelin basic protein (MBP), c-Jun and GAPDH asloading control (n=3), FIG. 4C is an immunoblot of macrophage number inthe nerve tissue for receptor tyrosine kinase ErbB2, Akt, phospho-Akt(pAkt), macrophage marker Iba-1 and GAPDH as loading control (n=3);

FIG. 5A is a schematic representation of the protocol used to assess theefficacy of rhNRG1 treatment on pre-existing schwannomas, FIG. 5B is aplot of maximum sciatic nerve diameters 3 and 6 months after nerve crushinjury for vehicles and rhNRG1-treated animals;

FIG. 6A is a plot of heart-body ratio after 13 weeks of treatment forvehicles and rhNRG1-treated animals, FIG. 6B are micrographs of sciaticnerve visualization in situ 3 months after sciatic nerve crush forvehicles and rhNRG1-treated animals;

FIG. 7 is a plot of myelin thickness in rhNRG1 treated and controlanimals as a functrion of frequency;

FIG. 8A is a schematic study protocol used to assess the efficacy ofsystemic rhNRG1 treatment on tumorlet burden in Nf2flox;Postn-Creanimals, FIG. 8B are micrographs of longitudinal nerve section fromsix-month-old mutant Nf2flox;Postn-Cre mice indicating a circumscribedarea of neoplastic Schwann cell proliferation (tumorlet), where thescale bars represent 50 μm, FIG. 8C is a plot of fraction of tumorlettissue within total tissue in relation to the total nerve area ininvestigated nerve sections with intraperitoneal saline injections (KOvehicle ip) compared to mutants on rhNRG1 injections (KO rhNRG1 ip); and

FIG. 9A is a study protocol used to assess the efficacy of systemicrhNRG1 treatment on schwannoma growth in Nf1flox;Postn-Cre animals, FIG.9B are micrographs of longitudinal nerve section from six-month-oldmutant Nf1flox;Postn-Cre mice indicating a circumscribed area ofneurofibroma growth, where the scale bars represent 50 μm, FIG. 9C is aplot of tumor tissue within total tissue for intraperitoneal salineinjections (KO vehicle ip) and rhNRG1 injections (KO rhNRG1 ip), FIG. 9Dis an immunoblot of pooled tissue from sciatic nerves, brachial nerves,trigeminal nerves and dorsal root ganglions (DRG) with either vehiclecontrol injections (KO vehicle ip) or rhNRG1 treatment (KO rhNRG1 ip)over 3 months for the listed immunostains.

DETAILED DESCRIPTION OF THE INVENTION

In Table 1 the nucleotide sequence of preferred embodiments ofneuregulins are listed.

TABLE 1 Nucleotide sequences of preferred neuregulin proteinsSEQ ID NO 1: ATGGGGAAAGGACGCGCGGGCCGAGTTGGCACCACA DNAGCCTTGCCTCCCCGATTGAAAGAGATGAAAAGCCAGG sequenceAATCGGCTGCAGGTTCCAAACTAGTCCTTCGGTGTGA of humanAACCAGTTCTGAATACTCCTCTCTCAGATTCAAGTGGT neuregulinTCAAGAATGGGAATGAATTGAATCGAAAAAACAAACC (NRG1),ACAAAATATCAAGATACAAAAAAAGCCAGGGAAGTC transcriptAGAACTTCGCATTAACAAAGCATCACTGGCTGATTCT variantGGAGAGTATATGTGCAAAGTGATCAGCAAATTAGGAA HRG-beta1c;ATGACAGTGCCTCTGCCAATATCACCATCGTGGAATC CDSAAACGAGATCATCACTGGTATGCCAGCCTCAACTGAA (92-1915)GGAGCATATGTGTCTTCAGCTACATCTACATCCACCAC of NCBITGGGACAAGCCATCTTGTAAAATGTGCGGAGAAGGAG referenceAAAACTTTCTGTGTGAATGGAGGGGAGTGCTTCATGG sequenceTGAAAGACCTTTCAAACCCCTCGAGATACTTGTGCAA NM_GTGCCCAAATGAGTTTACTGGTGATCGCTGCCAAAAC 001159995.1TACGTAATGGCCAGCTTCTACAAGCATCTTGGGATTGAATTTATGGAGGCGGAGGAGCTGTACCAGAAGAGAGTGCTGACCATAACCGGCATCTGCATCGCCCTCCTTGTGGTCGGCATCATGTGTGTGGTGGCCTACTGCAAAACCAAGAAACAGCGGAAAAAGCTGCATGACCGTCTTCGGCAGAGCCTTCGGTCTGAACGAAACAATATGATGAACATTGCCAATGGGCCTCACCATCCTAACCCACCCCCCGAGAATGTCCAGCTGGTGAATCAATACGTATCTAAAAACGTCATCTCCAGTGAGCATATTGTTGAGAGAGAAGCAGAGACATCCTTTTCCACCAGTCACTATACTTCCACAGCCCATCACTCCACTACTGTCACCCAGACTCCTAGCCACAGCTGGAGCAACGGACACACTGAAAGCATCCTTTCCGAAAGCCACTCTGTAATCGTGATGTCATCCGTAGAAAACAGTAGGCACAGCAGCCCAACTGGGGGCCCAAGAGGACGTCTTAATGGCACAGGAGGCCCTCGTGAATGTAACAGCTTCCTCAGGCATGCCAGAGAAACCCCTGATTCCTACCGAGACTCTCCTCATAGTGAAAGGTATGTGTCAGCCATGACCACCCCGGCTCGTATGTCACCTGTAGATTTCCACACGCCAAGCTCCCCCAAATCGCCCCCTTCGGAAATGTCTCCACCCGTGTCCAGCATGACGGTGTCCATGCCTTCCATGGCGGTCAGCCCCTTCATGGAAGAAGAGAGACCTCTACTTCTCGTGACACCACCAAGGCTGCGGGAGAAGAAGTTTGACCATCACCCTCAGCAGTTCAGCTCCTTCCACCACAACCCCGCGCATGACAGTAACAGCCTCCCTGCTAGCCCCTTGAGGATAGTGGAGGATGAGGAGTATGAAACGACCCAAGAGTACGAGCCAGCCCAAGAGCCTGTTAAGAAACTCGCCAATAGCCGGCGGGCCAAAAGAACCAAGCCCAATGGCCACATTGCTAACAGATTGGAAGTGGACAGCAACACAAGCTCCCAGAGCAGTAACTCAGAGAGTGAAACAGAAGATGAAAGAGTAGGTGAAGATACGCCTTTCCTGGGCATACAGAACCCCCTGGCAGCCAGTCTTGAGGCAACACCTGCCTTCCGCCTGGCTGACAGCAGGACTAACCCAGCAGGCCGCTTCTCGACACAGGAAGAAATCCAGGCCAGGCTGTCTAGTGTAATTGCTAACCAAGACCCTATTGCTGTA TAA SEQ ID NO 2:ATGGGGAAAGGACGCGCGGGCCGAGTTGGCACCACA DNA GCCTTGCCTCCCCGATTGAAAGAGATGAAAAGCCAGG sequenceAATCGGCTGCAGGTTCCAAACTAGTCCTTCGGTGTGA of humanAACCAGTTCTGAATACTCCTCTCTCAGATTCAAGTGGT neuregulinTCAAGAATGGGAATGAATTGAATCGAAAAAACAAACC (NRG1),ACAAAATATCAAGATACAAAAAAAGCCAGGGAAGTC transcriptAGAACTTCGCATTAACAAAGCATCACTGGCTGATTCT variantGGAGAGTATATGTGCAAAGTGATCAGCAAATTAGGAA HRG-beta1b;ATGACAGTGCCTCTGCCAATATCACCATCGTGGAATC CDSAAACGAGATCATCACTGGTATGCCAGCCTCAACTGAA (92-1966)GGAGCATATGTGTCTTCAGAGTCTCCCATTAGAATATC of NCBIAGTATCCACAGAAGGAGCAAATACTTCTTCATCTACA referenceTCTACATCCACCACTGGGACAAGCCATCTTGTAAAAT sequenceGTGCGGAGAAGGAGAAAACTTTCTGTGTGAATGGAGG NM_GGAGTGCTTCATGGTGAAAGACCTTTCAAACCCCTCG 001159999.1AGATACTTGTGCAAGTGCCCAAATGAGTTTACTGGTGATCGCTGCCAAAACTACGTAATGGCCAGCTTCTACAAGCATCTTGGGATTGAATTTATGGAGGCGGAGGAGCTGTACCAGAAGAGAGTGCTGACCATAACCGGCATCTGCATCGCCCTCCTTGTGGTCGGCATCATGTGTGTGGTGGCCTACTGCAAAACCAAGAAACAGCGGAAAAAGCTGCATGACCGTCTTCGGCAGAGCCTTCGGTCTGAACGAAACAATATGATGAACATTGCCAATGGGCCTCACCATCCTAACCCACCCCCCGAGAATGTCCAGCTGGTGAATCAATACGTATCTAAAAACGTCATCTCCAGTGAGCATATTGTTGAGAGAGAAGCAGAGACATCCTTTTCCACCAGTCACTATACTTCCACAGCCCATCACTCCACTACTGTCACCCAGACTCCTAGCCACAGCTGGAGCAACGGACACACTGAAAGCATCCTTTCCGAAAGCCACTCTGTAATCGTGATGTCATCCGTAGAAAACAGTAGGCACAGCAGCCCAACTGGGGGCCCAAGAGGACGTCTTAATGGCACAGGAGGCCCTCGTGAATGTAACAGCTTCCTCAGGCATGCCAGAGAAACCCCTGATTCCTACCGAGACTCTCCTCATAGTGAAAGGTATGTGTCAGCCATGACCACCCCGGCTCGTATGTCACCTGTAGATTTCCACACGCCAAGCTCCCCCAAATCGCCCCCTTCGGAAATGTCTCCACCCGTGTCCAGCATGACGGTGTCCATGCCTTCCATGGCGGTCAGCCCCTTCATGGAAGAAGAGAGACCTCTACTTCTCGTGACACCACCAAGGCTGCGGGAGAAGAAGTTTGACCATCACCCTCAGCAGTTCAGCTCCTTCCACCACAACCCCGCGCATGACAGTAACAGCCTCCCTGCTAGCCCCTTGAGGATAGTGGAGGATGAGGAGTATGAAACGACCCAAGAGTACGAGCCAGCCCAAGAGCCTGTTAAGAAACTCGCCAATAGCCGGCGGGCCAAAAGAACCAAGCCCAATGGCCACATTGCTAACAGATTGGAAGTGGACAGCAACACAAGCTCCCAGAGCAGTAACTCAGAGAGTGAAACAGAAGATGAAAGAGTAGGTGAAGATACGCCTTTCCTGGGCATACAGAACCCCCTGGCAGCCAGTCTTGAGGCAACACCTGCCTTCCGCCTGGCTGACAGCAGGACTAACCCAGCAGGCCGCTTCTCGACACAGGAAGAAATCCAGGCCAGGCTGTCTAGTGTAATTGCTAAC CAAGACCCTATTGCTGTATAA SEQ ID NO 3:ATGTCCGAGCGCAAAGAAGGCAGAGGCAAAGGGAAG DNAGGCAAGAAGAAGGAGCGAGGCTCCGGCAAGAAGCCG sequenceGAGTCCGCGGCGGGCAGCCAGAGCCCAGCCTTGCCTC ofCCCGATTGAAAGAGATGAAAAGCCAGGAATCGGCTGC human pro-AGGTTCCAAACTAGTCCTTCGGTGTGAAACCAGTTCTG neuregulin-1,AATACTCCTCTCTCAGATTCAAGTGGTTCAAGAATGGG membrane-AATGAATTGAATCGAAAAAACAAACCACAAAATATCA boundAGATACAAAAAAAGCCAGGGAAGTCAGAACTTCGCAT isoform TAACAAAGCATCACTGGCTGATTCTGGAGAGTATATG HRG-alpha;TGCAAAGTGATCAGCAAATTAGGAAATGACAGTGCCT CDSCTGCCAATATCACCATCGTGGAATCAAACGAGATCAT (518-2440)CACTGGTATGCCAGCCTCAACTGAAGGAGCATATGTG of NCBITCTTCAGAGTCTCCCATTAGAATATCAGTATCCACAGA referenceAGGAGCAAATACTTCTTCATCTACATCTACATCCACCA sequenceCTGGGACAAGCCATCTTGTAAAATGTGCGGAGAAGGA NM_GAAAACTTTCTGTGTGAATGGAGGGGAGTGCTTCATG 013964.3GTGAAAGACCTTTCAAACCCCTCGAGATACTTGTGCAAGTGCCAACCTGGATTCACTGGAGCAAGATGTACTGAGAATGTGCCCATGAAAGTCCAAAACCAAGAAAAGGCGGAGGAGCTGTACCAGAAGAGAGTGCTGACCATAACCGGCATCTGCATCGCCCTCCTTGTGGTCGGCATCATGTGTGTGGTGGCCTACTGCAAAACCAAGAAACAGCGGAAAAAGCTGCATGACCGTCTTCGGCAGAGCCTTCGGTCTGAACGAAACAATATGATGAACATTGCCAATGGGCCTCACCATCCTAACCCACCCCCCGAGAATGTCCAGCTGGTGAATCAATACGTATCTAAAAACGTCATCTCCAGTGAGCATATTGTTGAGAGAGAAGCAGAGACATCCTTTTCCACCAGTCACTATACTTCCACAGCCCATCACTCCACTACTGTCACCCAGACTCCTAGCCACAGCTGGAGCAACGGACACACTGAAAGCATCCTTTCCGAAAGCCACTCTGTAATCGTGATGTCATCCGTAGAAAACAGTAGGCACAGCAGCCCAACTGGGGGCCCAAGAGGACGTCTTAATGGCACAGGAGGCCCTCGTGAATGTAACAGCTTCCTCAGGCATGCCAGAGAAACCCCTGATTCCTACCGAGACTCTCCTCATAGTGAAAGGTATGTGTCAGCCATGACCACCCCGGCTCGTATGTCACCTGTAGATTTCCACACGCCAAGCTCCCCCAAATCGCCCCCTTCGGAAATGTCTCCACCCGTGTCCAGCATGACGGTGTCCATGCCTTCCATGGCGGTCAGCCCCTTCATGGAAGAAGAGAGACCTCTACTTCTCGTGACACCACCAAGGCTGCGGGAGAAGAAGTTTGACCATCACCCTCAGCAGTTCAGCTCCTTCCACCACAACCCCGCGCATGACAGTAACAGCCTCCCTGCTAGCCCCTTGAGGATAGTGGAGGATGAGGAGTATGAAACGACCCAAGAGTACGAGCCAGCCCAAGAGCCTGTTAAGAAACTCGCCAATAGCCGGCGGGCCAAAAGAACCAAGCCCAATGGCCACATTGCTAACAGATTGGAAGTGGACAGCAACACAAGCTCCCAGAGCAGTAACTCAGAGAGTGAAACAGAAGATGAAAGAGTAGGTGAAGATACGCCTTTCCTGGGCATACAGAACCCCCTGGCAGCCAGTCTTGAGGCAACACCTGCCTTCCGCCTGGCTGACAGCAGGACTAACCCAGCAGGCCGCTTCTCGACACAGGAAGAAATCCAGGCCAGGCTGTCTAGTGTAA TTGCTAACCAAGACCCTATTGCTGTATAASEQ ID NO 4: ATGTCCGAGCGCAAAGAAGGCAGAGGCAAAGGGAAG DNAGGCAAGAAGAAGGAGCGAGGCTCCGGCAAGAAGCCG sequence ofGAGTCCGCGGCGGGCAGCCAGAGCCCAGCCTTGCCTC human pro-CCCGATTGAAAGAGATGAAAAGCCAGGAATCGGCTGC neuregulin-1,AGGTTCCAAACTAGTCCTTCGGTGTGAAACCAGTTCTG membrane-AATACTCCTCTCTCAGATTCAAGTGGTTCAAGAATGGG bound AATGAATTGAATCGAAAAAACAAACCACAAAATATCA isoformAGATACAAAAAAAGCCAGGGAAGTCAGAACTTCGCAT HRG-gamma; TAACAAAGCATCACTGGCTGATTCTGGAGAGTATATG CDSTGCAAAGTGATCAGCAAATTAGGAAATGACAGTGCCT (518-1153)CTGCCAATATCACCATCGTGGAATCAAACGAGATCAT of NCBICACTGGTATGCCAGCCTCAACTGAAGGAGCATATGTG referenceTCTTCAGAGTCTCCCATTAGAATATCAGTATCCACAGA sequenceAGGAGCAAATACTTCTTCATCTACATCTACATCCACCA NM_CTGGGACAAGCCATCTTGTAAAATGTGCGGAGAAGGA 004495.3GAAAACTTTCTGTGTGAATGGAGGGGAGTGCTTCATGGTGAAAGACCTTTCAAACCCCTCGAGATACTTGTGCA AGTAA

In one embodiment the invention therefore encompasses a nucleic acidmolecule, and various uses thereof as described herein, selected fromthe group consisting of:

a) a nucleic acid molecule comprising or consisting of a nucleotidesequence that encodes an EGF-like domain of a neuregulin protein,preferably an EGF-like domain of a sequence according to SEQ ID NO 1-4,

b) a nucleic acid molecule comprising or consisting of a nucleotidesequence that encodes an EGF-like domain of a neuregulin protein,preferably the EGF-like domain of a sequence according to SEQ ID NO 1-4,wherein the length of the nucleic acid molecule is in between 50 and 500nucleic acids preferably between 100 and 300 nucleic acids, mostpreferably between 150 and 220 nucleic acids, wherein the surroundingsequences are preferably provided as neuregulin-encoding nucleotidesequences flanking the EGF-like domain,

c) a nucleic acid molecule which is complementary to a nucleotidesequence in accordance with a) or b);

d) a nucleic acid molecule comprising a nucleotide sequence havingsufficient sequence identity to be functionally analogous/equivalent toa nucleotide sequence according to a), b) or c), comprising preferably asequence identity to a nucleotide sequence according to a) or b) of atleast 70%, 80%, preferably 90%, more preferably 95%;

e) a nucleic acid molecule which, as a consequence of the genetic code,is degenerated into a nucleotide sequence according to a) through d);and

f) a nucleic acid molecule according to a nucleotide sequence of a)through e) which is modified by deletions, additions, substitutions,translocations, inversions and/or insertions and functionallyanalogous/equivalent to a nucleotide sequence according to a) throughd).

Further sequence variants are hereby incorporated in the invention thatexhibit an alternative nucleic acid sequence to SEQ ID NO 1-4 but encodethe same or a corresponding or functionally analogous amino acidsequence. Sequence variants obtained via using degeneracy of the geneticcode are included. Sequence optimized nucleic acid sequences of thosesequences provided herein are also included within the scope of theinvention.

Amino acid sequences of preferred neuregulin proteins are listed underTable 2.

TABLE 2 Amino acid sequences of preferred neuregulin proteinsSEQ ID NO 5: MGKGRAGRVGTTALPPRLKEMKSQESAAGSKLVLRCETS Amino acidSEYSSLRFKWFKNGNELNRKNKPQNIKIQKKPGKSELRIN sequence ofKASLADSGEYMCKVISKLGNDSASANITIVESNEIITGMPA humanSTEGAYVSSATSTSTTGTSHLVKCAEKEKTFCVNGGECF neuregulin (NRG1),MVKDLSNPSRYLCKCPNEFTGDRCONYVMASFYKHLGIE transcriptFMEAEELYQKRVLTITGICIALLVVGIMCVVAYCKTKKQR variantKKLHDRLRQSLRSERNNMMNIANGPHHPNPPPENVQLVN HRG-beta1c;QYVSKNVISSEHIVEREAETSFSTSHYTSTAHHSTTVTQTP NCBI ReferenceSHSWSNGHTESILSESHSVIVMSSVENSRHSSPTGGPRGRL Sequence:NGTGGPRECNSFLRHARETPDSYRDSPHSERYVSAMTTP NP_001153467.1ARMSPVDFHTPSSPKSPPSEMSPPVSSMTVSMPSMAVSPF The EGF-likeMEEERPLLLVTPPRLREKKFDHHPQQFSSFHHNPAHDSNS domain hasLPASPLRIVEDEEYETTQEYEPAQEPVKKLANSRRAKRTK been underlinedPNGHIANRLEVDSNTSSQSSNSESETEDERVGEDTPFLGIQNPLAASLEATPAFRLADSRTNPAGRFSTQEEIQARLSSVIA NQDPIAV SEQ ID NO 6:MGKGRAGRVGTTALPPRLKEMKSQESAAGSKLVLRCETS Amino acidSEYSSLRFKWFKNGNELNRKNKPQNIKIQKKPGKSELRIN sequence ofKASLADSGEYMCKVISKLGNDSASANITIVESNEIITGMPA human pro-STEGAYVSSESPIRISVSTEGANTSSSTSTSTTGTSHLVKCA neuregulin-1,EKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQ membrane-NYVMASFYKHLGIEFMEAEELYOKRVLTITGICIALLVVG bound isoformIMCVVAYCKTKKQRKKLHDRLRQSLRSERNNMMNIANG HRG-beta1b;PHHPNPPPENVQLVNQYVSKNVISSEHIVEREAETSFSTSH NCBI ReferenceYTSTAHHSTTVTQTPSHSWSNGHTESILSESHSVIVMSSVE Sequence:NSRHSSPTGGPRGRLNGTGGPRECNSFLRHARETPDSYRD NP_001153471.1SPHSERYVSAMTTPARMSPVDFHTPSSPKSPPSEMSPPVSS The EGF-likeMTVSMPSMAVSPFMEEERPLLLVTPPRLREKKFDHHPQQ domain hasFSSFHHNPAHDSNSLPASPLRIVEDEEYETTQEYEPAQEPV been underlinedKKLANSRRAKRTKPNGHIANRLEVDSNTSSQSSNSESETEDERVGEDTPFLGIQNPLAASLEATPAFRLADSRTNPAGRF STQEEIQARLSSVIANQDPIAVSEQ ID NO 7: MSERKEGRGKGKGKKKERGSGKKPESAAGSQSPALPPRL Amino acidKEMKSQESAAGSKLVLRCETSSEYSSLRFKWFKNGNELN sequence of RKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKL human pro-GNDSASANITIVESNEIITGMPASTEGAYVSSESPIRISVSTE neuregulin-1, GANTSSSTSTSTTGTSHLVKCAEKEKTFCVNGGECFMVK membrane-DLSNPSRYLCKCQPGFTGARCTENVPMKVONOEKAEELY bound isoformQKRVLTITGICIALLVVGIMCVVAYCKTKKQRKKLHDRL HRG-alphaRQSLRSERNNMMNIANGPHHPNPPPENVQLVNQYVSKN NCBI ReferenceVISSEHIVEREAETSFSTSHYTSTAHHSTTVTQTPSHSWSN Sequence:GHTESILSESHSVIVMSSVENSRHSSPTGGPRGRLNGTGGP NP_039258.1RECNSFLRHARETPDSYRDSPHSERYVSAMTTPARMSPV The EGF-like DFHTPSSPKSPPSEMSPPVSSMTVSMPSMAVSPFMEEERPL domain hasLLVTPPRLREKKFDHHPQQFSSFHHNPAHDSNSLPASPLRI been underlinedVEDEEYETTQEYEPAQEPVKKLANSRRAKRTKPNGHIANRLEVDSNTSSQSSNSESETEDERVGEDTPFLGIQNPLAASLEATPAFRLADSRTNPAGRFSTQEEIQARLSSVIANQDPIAV SEQ ID NO 8:MSERKEGRGKGKGKKKERGSGKKPESAAGSQSPALPPRL Human pro-KEMKSQESAAGSKLVLRCETSSEYSSLRFKWFKNGNELN neuregulin-1, RKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKL membrane-GNDSASANITIVESNEIITGMPASTEGAYVSSESPIRISVSTE bound isoform GANTSSSTSTSTTGTSHLVKCAEKEKTFCVNGGECFMVK HRG-gamma DLSNPSRYLCKcomprising an EGF-like domain (underlined) NCBI Reference Sequence:NP_004486.2

In one embodiment the invention therefore encompasses a polypeptide asdescribed herein comprising or consisting of an amino acid sequenceselected from the group consisting of:

-   -   a) an amino acid sequence comprising or consisting of the        EGF-like domain of a neuregulin protein, preferably the EGF-like        domain of an amino acid sequence according to SEQ ID NO 5-8,    -   b) an amino acid sequence comprising or consisting of the        EGF-like domain of a neuregulin protein, preferably the EGF-like        domain of an amino acid sequence according to SEQ ID NO 5-8,        wherein the length of the amino acid molecule is in between 25        and 150 amino acids preferably between 40 and 100 amino acids,        most preferably between 50 and 70 amino acids, wherein the        surrounding sequences are preferably provided as neuregulin        protein sequences flanking the EGF-like domain,    -   c) an amino acid sequence having sufficient sequence identity to        be functionally analogous/equivalent to an amino acid sequence        according to a), comprising preferably a sequence identity to an        amino acid sequence according to a) of at least 70%, 80%,        preferably 90%, more preferably 95%; and    -   d) an amino acid sequence of a), b) or c) which is modified by        deletions, additions, substitutions, translocations, inversions        and/or insertions and functionally analogous/equivalent to an        amino acid sequence according to a), b) or c).

The amino acid sequence of particularly preferred embodiments comprisingthe EGF-like domain of neuregulins as encompassed by the invention arelisted in Table 3:

TABLE 3 Preferred polypeptides comprising theEGF-like domains of a neuregulin protein SEQ ID NO 9:SHLVKCAEKEKTFCVNGGECFM Amino acid VKDLSNPSRYLCKCPNEFTGDR sequence ofCQNYVMASFYKHLGIEF recombinant human neuregulin-1 betaEGF domain (rhNGR1) Obtainable from transcript variant HRG-beta1c;NP_001153467.1 SEQ ID NO 10: SHLVKCAEKEKTFCVNGGECFM amino acidVKDLSNPSRYLCKCQPGFTGAR sequence of  CT the EGF-like domain of humanpro-neuregulin-1, membrane- bound isoform HRG-alpha; NP_039258.1SEQ ID NO 11: SHLVKCAEKEKTFCVNGGECFM amino acid VKDLSNPSRYLCKsequence of  the EGF-like domain of human neuregulin-1, membrane-bound isoform HRG-gamma; NP_004486.2 SEQ ID NO 12:CVNGGECFMVKDLSNPSRYLCK CPNEFTGDRCQ SEQ ID NO 13: CVNGGECFMVKDLSNPSRYLCKCQPGFTGARCT SEQ ID NO 14: CVNGGECFMVKDLSNPSRYLCK

According to the present invention the term “EGF-like domain of aneuregulin protein” encompasses the sequences identified under SEQ ID NO9-14, including sequence variants thereof such as are described herein,and functionally analogous sequences.

In some embodiments the “EGF-like domain of a neuregulin protein”preferably refers to a polypeptide encoded by the neuregulin gene thatbinds to and activates ErbB2, ErbB3, ErbB4 or combinations thereof andbears a structural and/or sequence similarity to the polypeptideaccording to SEQ ID NO 9. The term “EGF-like domain of a neuregulinprotein” therefore also encompasses any protein sequences withessentially the same or similar activity as SEQ ID NO 9, when tested forthe binding to and activation of ErbB2, ErbB3 or ErbB4, whereinessentially the same or similar activity relates to at least 5%,preferably 10, 20, 30, 50 or more preferably at least 70% of theactivity of SEQ ID NO 9.

A preferred embodiment of the invention therefore encompasses apolypeptide as described herein comprising or consisting of an aminoacid sequence selected from the group consisting of:

a) an amino acid sequence according to SEQ ID NO 9-14

b) an amino acid sequence according to a) that comprises a 0 to 10 aminoacid addition or deletion at the N and/or C terminus,

c) an amino acid sequence comprising an amino acid sequence according toto a) or b), wherein the length of the amino acid sequence is in between25 and 150 amino acids preferably between 40 and 100 amino acids, mostpreferably between 50 and 70 amino acids, amino acids, wherein thesurrounding sequences are preferably provided as neuregulin proteinsequences flanking the EGF-like domain, such as derived from SEQ ID NO5-8,

d) an amino acid sequence having sufficient sequence identity to befunctionally analogous/equivalent to an amino acid sequence according toa), b) or c), comprising preferably a sequence identity to an amino acidsequence according to a) of at least 70%, 80%, preferably 90%, morepreferably 95%; and

e) an amino acid molecule according to an amino acid sequence of a), b),c) or d) which is modified by deletions, additions, substitutions,translocations, inversions and/or insertions and functionallyanalogous/equivalent to an amino acid sequence according to a), b), c)or d).

Functionally analogous sequences refer preferably to the ability toencode a functional peptide comprising the EGF-like domain.

Protein modifications to the polypeptide comprising the EGF-like domain,which may occur through substitutions in amino acid sequence, andnucleic acid sequences encoding such molecules, are also included withinthe scope of the invention. Substitutions as defined herein aremodifications made to the amino acid sequence of the protein, wherebyone or more amino acids are replaced with the same number of (different)amino acids, producing a protein which contains a different amino acidsequence than the primary protein. In some embodiments this amendmentwill not significantly alter the function of the protein. Likeadditions, substitutions may be natural or artificial. It is well knownin the art that amino acid substitutions may be made withoutsignificantly altering the protein's function. This is particularly truewhen the modification relates to a “conservative” amino acidsubstitution, which is the substitution of one amino acid for another ofsimilar properties. Such “conserved” amino acids can be natural orsynthetic amino acids which because of size, charge, polarity andconformation can be substituted without significantly affecting thestructure and function of the protein. Frequently, many amino acids maybe substituted by conservative amino acids without deleteriouslyaffecting the protein's function.

In general, the non-polar amino acids Gly, Ala, Val, lie and Leu; thenon-polar aromatic amino acids Phe, Trp and Tyr; the neutral polar aminoacids Ser, Thr, Cys, Gin, Asn and Met; the positively charged aminoacids Lys, Arg and His; the negatively charged amino acids Asp and Glu,represent groups of conservative amino acids. This list is notexhaustive. For example, it is well known that Ala, Gly, Ser andsometimes Cys can substitute for each other even though they belong todifferent groups.

The present invention encompasses gene therapy comprising theadministration of a therapeutic gene encoding the neuregulins andEGF-like domains described herein.

The term gene therapy preferably refers to the transfer of DNA into asubject in order to treat a disease. The person skilled in the art knowsstrategies to perform gene therapy using gene therapy vectors. Such genetherapy vectors are optimized to deliver foreign DNA into the host cellsof the subject. In a preferred embodiment the gene therapy vectors maybe a viral vector. Viruses have naturally developed strategies toincorporate DNA in to the genome of host cells and may therefore beadvantageously used. Preferred viral gene therapy vectors may includebut are not limited to retroviral vectors such as moloney murineleukemia virus (MMLV), adenoviral vectors, lentiviral,adenovirus-associated viral (AAV) vectors, pox virus vectors, herpessimplex virus vectors or human immunodeficiency virus vectors (HIV-1).However also non-viral vectors may be preferably used for the genetherapy such as plasmid DNA expression vectors driven by eukaryoticpromoters or liposomes encapsulating the transfer DNA. Furthermorepreferred gene therapy vectors may also refer to methods to transfer ofthe DNA such as electroporation or direct injection of nucleic acidsinto the subject. Moreover it may be preferred that the gene therapyvectors for example a viral gene therapy vector is adapted to targettumour cells and in particular tumour cells of the nervous system. Tothis end the viral capsid may be conjugated with ligands binding totumour cells such as epidermal growth factors, basic fibroblast growthvectors or monoclonal antibodies. It may also be preferred that theviral gene therapy vectors are genetically modified using tumourspecific promoters to enhance the expression of the nucleic acidspecifically within the tumour cells. Preferred gene therapy vectors maytherefore comprise vectors for an inducible or conditional expression ofthe polypeptides. The person skilled in the art knows how to choosepreferred gene therapy vectors according the need of application as wellas the methods on how to implement the nucleic acid into the genetherapy vector. (P. Seth et al., 2005, N. Koostra et, al. 2009., W.Walther et al. 2000, Waehler et al. 2007)

The nucleic acid according to the invention and preferred embodimentsthereof, in particular a nucleic acid encoding for a polypeptideaccording to SEQ ID 9, is particularly efficient for gene therapy due toa high therapeutic potential at a small size. This ensures a stableintegration at high expression levels over extended periods of times.The preferred embodiment of the gene therapy vector is not onlyefficient for the treatment of tumours of the nervous system, but alsofor an effective prevention of an outbreak of a tumour of the nervoussystem. It may therefore be particularly preferred to use the genetherapy vector as a preventive treatment for subjects afflicted by agenetic deficiency in neurofibromatosis type 1, neurofibromatosis type 2and/or neurofibromatosis type 3.

In a further preferred embodiment the invention relates to a cell foruse as a medicament in the treatment and/or prevention of a tumour ofthe nervous system as described herein, wherein the cell is geneticallymodified and comprises an exogenous nucleic acid region encoding for apolypeptide according to the invention or preferred embodiments thereofand wherein the exogenous nucleic acid region is operably linked to apromoter.

The person skilled in the art knows how to genetically modify cells inorder to express the polypeptides according to the inventions.Advantageously by expressing the therapeutically effective polypeptidesthe cells may act as bio pump or drug factory that continuouslyexpresses and provides the polypeptides to the subject. Thereby theamount of the polypeptides can be held at a therapeutic level over longperiods. The person skilled in the art knows which cells may bepreferably used to this end. In a preferred embodiment the cells arestem cells, characterized by a stable expression of the polypeptides.Stem cells may include but are not limited to, embryonic stem cells suchas early embryonic stem cells and blastocyst embryonic stem cells; fetalstem cells; umbilical cord stem cells; and adult stem cells such asmesenchymal stem cells, hematopoietic stem cells, endothelial stemcells, peripheral blood stem cells, and multipotent somatic stem cells.

The cells may migrate to the site of the tumour in order to locallyexpress the polypeptides in vicinity of the tumour. Advantageously thecells may however also be transplanted at a different location as thepolypeptides can also be transported by the vascular system throughoutthe body of the subject. Local administration of the cells e.g. by asubcutaneous injection may therefore contribute in a systemic mannerlargely irrespective of the location of the cells within the body of thesubject.

In a further preferred embodiment the cells for use as a medicament asdescribed herein is characterised by introducing a therapeuticallyeffective number of said cells to a subject within a biocompatiblematrix. Preferred materials for the biocompatible matrix are agarose,carrageenan, alginate, chitosan, gellan gum, hyaluronic acid, collagen,cellulose and its derivatives, gelatin, elastin, epoxy resin, photocross-linkable resins, polyacrylamide, polyester, polystyrene andpolyurethane or polyethylene glycol (PEG). It is further preferred thatthe biocompatible matrix is a semi-permeable hydrogel matrix and thecells are entrapped by said matrix. Advantageously the biocompatiblematrix allows for an efficient diffusion of nutrients, oxygens and otherbiomolecules to ensure a long lasting viability of the cells expressingthe polypeptide, while immobilizing the cells. Thereby the cells can beconcentrated at preferred locations within the subject. For instance thecells can be transplanted subcutaneously and/or in proximity of diseasedregions of the subject i.e. close to a vestibular schwannoma. It issurprising that by introducing encapsulated cells, the cells functionparticularly efficiently as bio pumps and provide a high level oftherapeutic polypeptides to the subject.

In a preferred embodiment the invention further relate to pharmaceuticalcomposition for use as a medicament in the treatment and/or preventionof a tumour of the nervous system as described herein, wherein thepharmaceutical composition comprises the polypeptide, the nucleic acid,the gene therapy vector and/or the cell, and optionally apharmaceutically accepted carrier. Preferably the pharmaceuticalcomposition is administered to the subject at a therapeuticallyeffective amount at any administration route as described herein.

In a preferred embodiment the pharmaceutical composition for use as amedicament as described herein is administered by introducing atherapeutically effective amount of the composition into the bloodstream of a subject. This route of administration is particularlyadvantageous for an administration of the polypeptides. Advantageouslythe polypeptides and in particular the soluble polypeptides as describedherein can cross the blood-brain barrier. Therefore a systemicadministration by introducing a therapeutically effective amount of thepolypeptides into the vascular system may be used to treat tumours ofthe nervous system throughout the body of the subject including thebrain.

In a further preferred embodiment the pharmaceutical composition for useas a medicament as described herein is administered locally. It isparticularly preferred that the pharmaceutical composition isadministered locally to the site of the tumour of the nervous system.For instance in case of a skin tumour the pharmaceutical composition maybe administered subcutaneously in close proximity of the tumoroustissue. It may also be preferred that the local administration of thepharmaceutical composition to the skin is achieved by crèmes or lotionsthat comprise the polypeptides.

Moreover in a preferred embodiment the local administration of thepolypeptides may be preferably mediated by an implant such as a collagensponge. To this end it may be preferred to soak the sponge in apharmaceutical composition comprising the polypeptides and implant thesponge close to the site of the tumour. By doing so the polypeptidesadvantageously diffuse locally and can therefore site specific impedeand/or reverse the growth of the tumour.

In further preferred embodiment the polypeptides may be locallyadministered by means of a hydrogel. Hydrogels are three-dimensional,cross-linked networks of water-soluble polymers. The person skilled inthe art knows how to produce suitable hydrogels for the delivery ofproteins or polypeptides (Hoare et al. 2008, Peppas et al. 2000,Hoffmann A. et al. 2012). In particular the density of the cross-linkednetwork of the hydrogel may be advantageously optimized to achieve aporosity suited to load the polypeptides into the hydrogel. Subsequentlythe release of the polypeptides is governed by the diffusion of thepeptides throughout the gel network. Therefore the release rate and thusthe therapeutically effective amount of the polypeptides can beprecisely tuned by optimizing the cross-linking density of the hydrogel.Moreover preferred hydrogels may also encompass an outer membraneoptimized for the release of the polypeptides. The preferred hydrogelsare biocompatible and are preferably implanted for a long term localsupply of the polypeptides. In preferred embodiments the hydrogels maybe implanted subcutaneously at or close to the site of the tumour.Transdermal administration of the polypeptides by use of hydrogels mayalso be envisioned. By means hydrogels a therapeutically effective doseof polypeptides can be advantageously localized to the site of thetumour, while minimizing the systemic dosage. Thereby a long termadministration can be achieved with a sustained and site specificrelease and minimized side effects.

As used herein, “nucleic acid” shall mean any nucleic acid molecule,including, without limitation, DNA, RNA and hybrids or modified variantsthereof. An “exogenous nucleic acid” or “exogenous genetic element”relates to any nucleic acid introduced into the cell, which is not acomponent of the cells “original” or “natural” genome. Exogenous nucleicacids may be integrated or non-integrated, or relate to stablytransfected nucleic acids.

As used herein, “polypeptide” shall mean both peptides and proteins. Inthis invention, the polypeptides may be naturally occurring orrecombinant (i.e., produced via recombinant DNA technology), and maycontain mutations (e.g., point, insertion and deletion mutations) aswell as other covalent modifications (e.g., glycosylation and labelling(via biotin, streptavidin, fluorescein, and radioisotopes)) or othermolecular bonds to additional components. For example, PEGylate proteinsare encompassed by the scope of the present invention. PEGylation hasbeen widely used as a post-production modification methodology forimproving biomedical efficacy and physicochemical properties oftherapeutic proteins. Applicability and safety of this technology havebeen proven by use of various PEGylated pharmaceuticals for many years(refer Jevsevar et al, Biotechnol J. 2010 January; 5(1):113-28). In someembodiments the polypeptides described herein are modified to exhibitlonger in vivo half-lives and resist degradation when compared tounmodified polypeptides. Such modifications are known to a skilledperson, such as cyclized polypeptides, polypeptides fused to VitaminB12, stapled peptides, protein lipidization and the substitution ofnatural L-amino acids with D-amino acids (refer Bruno et al, Ther Deliv.2013 November; 4(11): 1443-1467).

As used herein the term “a 0 to 10 amino acid addition or deletion atthe N and/or C terminus of a sequence” means that the polypeptide mayhave a) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at itsN terminus and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted atits C terminus orb) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacids at its C terminus and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10nucleotides deleted at its N terminus, c) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 additional amino acids at its N terminus and 0, 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 additional amino acids at its N terminus or d) 0, 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 amino acids deleted at its N terminus and 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids deleted at its C terminus.

Furthermore, in addition to the polypeptides described herein,peptidomimetics are also contemplated. Peptide analogs are commonly usedin the pharmaceutical industry as non-peptide drugs with propertiesanalogous to those of the template peptide. These types of non-peptidecompound are termed “peptide mimetics” or “peptidomimetics” (Fauchere(1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p. 392;and Evans et al. (1987) J. Med. Chem. 30: 1229) and are usuallydeveloped with the aid of computerized molecular modelling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. It may be preferred in some embodiments to usepeptide mimetics in order to prolong the stability of the polypeptides,when administered to a subject. To this end peptide mimetics for thepolypeptides may be preferred that are not cleaved by human proteasomes.

The polypeptides, nucleic acid molecules, gene therapy vectors or cellsdescribed herein may comprise different types of carriers depending onwhether they are to be administered in solid, liquid or aerosol form,and whether they need to be sterile for such routes of administration asinjection.

The active agent present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, directly, via a catheter, viaa lavage, in cremes, in lipid compositions (e.g., liposomes), locallyapplied by sponges or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

The present invention encompasses treatment of a patient by introducinga therapeutically effective number polypeptides, nucleic acids, genetherapy vectors or cells into a subject's bloodstream. As used herein,“introducing” polypeptides, nucleic acids, gene therapy vectors or cellsinto the subject's bloodstream shall include, without limitation,introducing such polypeptides, nucleic acids, gene therapy vectors orcells into one of the subject's veins or arteries via injection. Suchadministering can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods. A single injection ispreferred, but repeated injections over time (e.g., quarterly,half-yearly or yearly) may be necessary in some instances. Suchadministering is also preferably performed using an admixture ofpolypeptides, nucleic acids, gene therapy vectors or cells and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known to those skilled in the art and include, but arenot limited to, 0.01-0.1 M and preferably 0.05 M phosphate buffer or0.8% saline.

Administration may also occur locally, for example by injection into anarea of the subject's body in proximity to a tumour disease. As usedherein, in “proximity with” a tissue includes, for example, within 50mm, 20 mm, 10 mm, 5 mm, within 1 mm of the tissue, within 0.5 mm of thetissue and within 0.25 mm of the tissue.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

Additionally, such pharmaceutically acceptable carriers can be aqueousor non-aqueous solutions, suspensions, and emulsions, most preferablyaqueous solutions. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions and suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as Ringer's dextrose, those based onRinger's dextrose, and the like. Fluids used commonly for i.v.administration are found, for example, in Remington: The Science andPractice of Pharmacy, 20th Ed., p. 808, Lippincott Williams S-Wilkins(2000). Preservatives and other additives may also be present, such as,for example, antimicrobials, antioxidants, chelating agents, inertgases, and the like.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

The composition can be formulated in a neutral or salt form.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike.

As used herein, a “therapeutically effective amount” for thepharmaceutical composition includes, without limitation, the followingamounts and ranges of amounts:

For a composition comprising a polypeptide according to the invention orpreferred embodiment thereof: (i) from about 1×10⁻³ to about 1×10⁶ μg/kgbody weight; (ii) from about 1×10⁻² to about 1×10⁵ μg/kg body weight;(iii) from about 1×10⁻¹ to about 1×10⁴ μg/kg body weight; (iv) fromabout 1×10⁻¹ to about 1×10³ μg/kg body weight; (v) from about 1×10⁻¹ toabout 1×10² μg/kg body weight; (vi) from about 1×10⁻¹ to about 0.5×10²μg/kg body weight; (vii) about 1×10⁻² μg/kg body weight; (viii) about1×10¹ μg/kg body weight; (ix) about 10 μg/kg body weight (x) about 1×10²μg/kg body weight; (xi) about 5×10³ μg/kg body weight.

For a composition comprising cells according to the invention orpreferred embodiment thereof: (i) from about 1×10² to about 1×10⁸cells/kg body weight; (ii) from about 1×10³ to about 1×10⁷ cells/kg bodyweight; (iii) from about 1×10⁴ to about 1×10⁶ cells/kg body weight; (iv)from about 1×10⁴ to about 1×10⁵ cells/kg body weight; (v) from about1×10⁵ to about 1×10⁶ cells/kg body weight; (vi) from about 5×10⁴ toabout 0.5×10⁵ cells/kg body weight; (vii) about 1×10³ cells/kg bodyweight; (viii) about 1×10⁴ cells/kg body weight; (ix) about 5×10⁴cells/kg body weight; (x) about 1×10⁵ cells/kg body weight; (xi) about5×10⁵ cells/kg body weight; (xii) about 1×10⁶ cells/kg body weight; and(xiii) about 1×10⁷ cells/kg body weight.

Human body weights envisioned include, without limitation, about 5 kg,10 kg, 15 kg, 30 kg, 50 kg, about 60 kg; about 70 kg; about 80 kg, about90 kg; about 100 kg, about 120 kg and about 150 kg.

Dosages of the viral gene therapy vector will depend primarily onfactors such as the condition being treated, the selected gene, the age,weight and health of the patient, and may thus vary among patients. Forexample, a therapeutically effective human dosage of the viral vectorsmay be preferably in the range of from about 1 to about 1000 ml,preferably 10 to 100 ml, preferably 20 to 50 ml of saline solutioncontaining concentrations of from about 1×10⁵ to 1×10¹² preferably 1×10⁶to 1×10¹¹ more preferably 1×10⁷ to 1×10¹⁰ plaque forming units (pfu)/mlviruses. The dosage will be adjusted to balance the therapeutic benefitagainst any side effects. The levels of expression of the selected genecan be monitored to determine the selection, adjustment or frequency ofdosage administration.

As used herein “inducible expression” or “conditional expression”relates to a state, multiple states or system of an expression of thepolypeptide, wherein the polypeptide is preferably not expressed, or insome embodiments expressed at negligible or relatively low levels,unless there is the presence of one or more molecules (an inducer) orother set of conditions in the cell that allows for polypeptideexpression. Inducible promoters may relate to either naturally occurringpromoters that are expressed at a relatively higher level underparticular biological conditions, or to other synthetic promoterscomprising any given inducible element. Inducible promoters may refer tothose induced by particular tissue- or micro-environments orcombinations of biological signals present in particular tissue- ormicro-environments, or to promoters induced by external factors, forexample by administration of a small drug molecule or other externallyapplied signal.

As used herein the term “bio pump” or “drug factory” preferably describethe function of cells as a continuously producing source of thepolypeptide, preferably at a therapeutically effective dosage. Byadministering cells to a subject particularly stable levels of thepolypeptides according to the invention or preferred embodiments thereofcan be provided. In the sense the bio pump, that is the cells, allow fora continuous supply that maintain levels of the polypeptides at aparticular high and stable state, for example it may compensate forlosses of the polypeptides for instance due to a degeneration of thepolypeptides through proteasomes.

The terms “hydrogel”, “gel” and the like, are preferably usedinterchangeably herein to refer to a material which is not a readilyflowable liquid and not a solid. The term hydrogel is preferably meantto be a water insoluble, water-containing material. Examples ofhydrogels include synthetic polymers such as polyhydroxyethylmethacrylate, poly(ethylene glycol) and chemically or physicallycrosslinked polyvinyl alcohol, polyacrylamide, poly(N-vinyl pyrolidone),polyethylene oxide, and hydrolysed polyacrylonitrile. Examples ofhydrogels which are organic polymers include DNA hydrogels or covalentor ionically crosslinked polysaccharide-based hydrogels such as thepolyvalent metal salts of alginate, pectin, carboxymethyl cellulose,heparin, hyaluronate and hydrogels from chitin, chitosan, pullulan,gellan and xanthan.

As used herein the term “tumour of the nervous system” refers to anabnormal growth and/or alternation of tissues or cells of the nervoussystem and refers thus to benign tumours or neoplasm as well asmalignant tumours or neoplasm (cancers) of the nervous system inparticular the central nervous system. Thus a tumour of the nervoussystem include in particular primary tumours of glial, neuronal, Schwanncell, pinealcyte, menningioma and melanoma, as well as sarcoma, lymphomaand multiple systemic malignancies that metastasize in the brain.

Specific examples of tumours of the nervous system include, but are notlimited to, astrocytoma, glioblastoma, oligodendroglioma, ependymoma,choroid plexus carcinoma, angiocentric glioma, ganglioglioma,neurocytoma, dysplastic neuroepithelial tumour, paranglioma of thespinal cord, pineocytoma, pineocytoma, primitive neuroectodermal tumour,ganglioneuroma, schwannoma, neurofibroma, perinerioma, malignantperipheral nerve sheath tumour, meningioma, or haemangioblastoma.

A therapeutically effective amount of composition for the treatment ofmedical conditions associated with the treatment and/or prevention ofmedical conditions associated neurofibromatosis (NF), thus a geneticdeficiency in neurofibromatosis type 1, neurofibromatosis type 2 and/orneurofibromatosis type 3 the therapeutically effective amount of thecomposition is preferably sufficient to achieve at least one, two,three, four or more of the following effects: (i) the reduction oramelioration of the severity of one or more NF symptoms; (ii) thereduction in the duration of one or more symptoms associated with NF;(iii) the prevention in the recurrence of a tumour or a symptomassociated with NF; (iv) the regression of tumours and/or one or moresymptoms associated therewith; (v) the reduction in hospitalization of asubject; (vi) the reduction in hospitalization length; (vii) theincrease in the survival of a subject; (viii) the inhibition of theprogression of tumours and/or one or more symptoms associated therewith;(ix) the enhancement or improvement of the therapeutic effect of anothertherapy; (x) a reduction or elimination of hearing loss, tinnitus,visual impairment, imbalance, or painful skin lesions associated withNF; (xi) a reduction in the growth of a tumour or neoplasm associatedwith NF; (xii) a decrease in tumour size (e.g., volume or diameter) ofneurofibromas, plexiform neurofibromas, and/or NF2-associated tumours(e.g., meningiomas, schwannomas, ependymomas, etc.); (xiii) a reductionin the formation of a newly formed tumour, for example an NF-associatedtumour such as neurofibromas, plexiform neurofibromas, or NF2-associatedtumours (e.g., meningiomas, schwannomas, ependymomas, etc.); (xiv)eradication, removal, or control of primary, regional and/or metastatictumours associated with NF; (xv) ease in removal of tumours by reducingvascularization prior to surgery; (xvi) a decrease in the number or sizeof metastases; (xvii) a reduction in mortality; (xviii) an increase inthe tumour-free survival rate of patients; (xix) an increase in relapsefree survival; (xx) an increase in the number of patients in remission;(xxi) a decrease in hospitalization rate; (xxii) the size of the tumouris maintained and does not increase or increases by less than theincrease of a tumour after administration of a standard therapy asmeasured by conventional methods available to one of skill in the art,such as magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI(DCE-MRI), X-ray, and computed tomography (CT) scan, or a positronemission tomography (PET) scan; (xxiii) the prevention of thedevelopment or onset of one or more symptoms associated with NF; (xxiv)an increase in the length of remission in patients; (xxv) the reductionin the number of symptoms associated with NF; (xxvi) an increase insymptom-free survival of NF patients; (xxvii) improvement in neuralfunction, e.g., hearing, balance, tinnitus, or vision; (xxvi) inhibitionor decrease in tumour metabolism or perfusion; (xxvii) inhibition ordecrease in angiogenesis or vascularization of the tumour; (xxviii)improvement in quality of life as assessed by methods well known in theart, e.g., tinnitus questionnaires; and/or (xxix) an improvement inhearing, hearing function, or word recognition.

As used herein, “treating” a subject afflicted with a disorder shallmean slowing, stopping or reversing the disorder's progression. In thepreferred embodiment, treating a subject afflicted with a disorder meansreversing the disorder's progression, ideally to the point ofeliminating the disorder itself. As used herein, ameliorating a disorderand treating a disorder are equivalent. The treatment of the presentinvention may also, or alternatively, relate to a prophylacticadministration of the active agents described herein. Such aprophylactic administration may relate to the prevention of any givenmedical disorder, or the prevention of development of said disorder,whereby prevention or prophylaxis is not to be construed narrowly underall conditions as absolute prevention. Prevention or prophylaxis mayalso relate to a reduction of the risk of a subject developing any givenmedical condition, preferably in a subject at risk of said condition.

DETAILED DESCRIPTION OF THE FIGURES

FIGS. 1A-C: Study design and rhNRG1 pharmacokinetics

FIG. 1A, Schematic representation of the protocol used to assess theefficacy of rhNRG1 treatment on schwannoma growth after experimentalsciatic nerve crush injury. FIG. 1B, Serum levels of rhNRG1 after singleintraperitoneal injections of 10 μg rhNRG1 per kg body weight measuredin pooled blood (n=4 animals; median). FIG. 1C, Body weight of animalsreceiving (genotype and treatment as indicated) was determined over 13weeks.

FIGS. 2A-D: Functional nerve regeneration after nerve crush

FIG. 2A-C, Foot-base angle (FBA) quantification ofNf2flox;PO-Cre;Nefh-Cre mutant mice receiving no treatment (no treatmentko), saline injections (vehicle ip ko) or intraperitoneal administrationof Neregulin1 (rhNRG1 ip ko). FBA baseline levels were measured beforenerve injury (week 0). Functional motor recovery after sciatic nervecrush was assessed for 8 consecutive weeks (* P<0.05 for differencesfrom non-treated animals; #P<0.05 for differences from baseline withinsame group; two-way ANOVA for repeated measures with Holm-Sidak post-hoctest; n=8 animals per genotype; mean±SEM). FIG. 2D, rhNRG1 release fromrhNRG1-soaked sponges in vitro was estimated over time as proteinconcentration within PBS solution following 24 hours of incubation.

FIGS. 3A-B: Sciatic nerve diameter following rhNRG1 treatment

FIG. 3A and FIG. 3B, Quantification of sciatic nerve diameters 3 monthsafter nerve crush injury. (A) Wildtype mice (WT vehicle) were comparedwith Nf2flox;PO-Cre;Nefh-Cre mutant mice receiving saline injections (KOvehicle) or intraperitoneal administration of Neregulin1 (KO rhNRG1;(*** P<0.001 for differences between vehicle and rhNRG1-treated animals;n=8 animals per genotype; mean±SEM). (FIG. 3B) Wildtype mice (WT vehicleACS) were compared with Nf2flox;PO-Cre;Nefh-Cre mutant mice receivingsaline loaded absorbable collagen sponges (KO vehicle ACS) or rhNRG1loaded sponges (KO rhNRG1 ACS; (*** P<0.001 for differences betweenvehicle and rhNRG1-treated animals; n=8 animals per genotype; mean±SEM).

FIGS. 4A-C: Signalling and neuropathological assessment

FIG. 4A, Sciatic nerve cross sections of indicated cohorts 3 monthsafter crush injury were either HE stained (a-f), or immunolabeled (browncolor), for Schwann cell markers S100 and MPZ, Neuregulin1, the receptortyrosine kinase ErbB2, p75 and CPI-17. Cell nuclei are visualized inblue. Scale bar represents 20 μm. FIG. 4B and FIG. 4C, Immunoblot ofsciatic nerve lysates (pooled tissue from at least three differentanimals per indicated genotype was prepared from crushed sciaticnerves). (FIG. 4B) Immunoblot for Erk1/2, phospho-Erk1/2 (pErk1/2),myelin protein zero (MPZ), myelin basic protein (MBP), c-Jun and GAPDHas loading control (n=3). (FIG. 4C) Immunoblot for receptor tyrosinekinase ErbB2, Akt, phospho-Akt (pAkt), macrophage marker Iba-1 and GAPDHas loading control (n=3).

FIGS. 5A-B: Pre-existing tumors

FIG. 5A, Schematic representation of the protocol used to assess theefficacy of rhNRG1 treatment on pre-existing schwannomas. FIG. 5B,Quantification of maximum sciatic nerve diameters 3 and 6 months afternerve crush injury. Nf2flox;PO-Cre;Nefh-Cre mutant mice received eithersaline injections (vehicle) or intraperitoneal administration of rhNRH1(** P<0.01 for differences between vehicle and rhNRG1-treated animals;n=6 animals per genotype; mean±SEM).

FIGS. 6A-B: Heart-body ratio and sciatic nerve visualization

FIG. 6A, The heart-body ratio was determined in animals (genotype andtreatment as indicated) after 13 weeks of treatment (n=8 animals pergenotype; mean±SEM). FIG. 6B, Sciatic nerve visualization in situ 3months after sciatic nerve crush. Representative images for assessmentof sciatic nerve diameter as indicator for tumour size from wildtypemice (rhNRG1 ip wt) and Nf2flox;PO-Cre;Nefh-Cre mutant mice receivingsaline injections (vehicle ip ko) or intraperitoneal administration ofNeregulin1 (rhNRG1 ip ko).

FIG. 7 : Myelin thickness

Frequency blot of myelinated axons calculated from sciatic nerve crosssections of 5-month-old wildtype mice receiving either systemic rhNRG1or vehicle treatment for 3 consecutive months.

FIGS. 8A-C: Tumorlet burden in nerves of Nf2flox;Postn-Cre mutant mice

FIG. 8A, Study protocol used to assess the efficacy of systemic rhNRG1treatment on tumorlet burden in Nf2flox;Postn-Cre animals. FIG. 8B,Representative longitudinal nerve section from six-month-old mutantNf2flox;Postn-Cre mice indicating a circumscribed area of neoplasticSchwann cell proliferation (tumorlet). Scale bars represent 50 μm. FIG.8C, Fraction of tumorlet tissue within total tissue was quantified asarea covered by tumorlets in relation to the total nerve area ininvestigated nerve sections. Nf2flox;Postn-Cre mutant mice receivingintraperitoneal saline injections (KO vehicle ip) were compared tomutants on rhNRG1 injections (KO rhNRG1 ip; n=6 animals per genotype;mean±SEM).

FIGS. 9A-D: Tumor burden in nerves of Nf1flox;Postn-Cre mutant mice

FIG. 9A, Study protocol used to assess the efficacy of systemic rhNRG1treatment on schwannoma growth in Nf1flox;Postn-Cre animals. FIG. 9B,Representative longitudinal nerve section from six-month-old mutantNf1flox;Postn-Cre mice indicating a circumscribed area of neurofibromagrowth. Scale bars represent 50 FIG. 9C, Fraction of tumor tissue withintotal tissue was quantified as area covered by tumor in relation to thetotal nerve area in investigated nerve sections. Nf1flox;Postn-Cremutant mice receiving intraperitoneal saline injections (KO vehicle ip)were compared to mutants on rhNRG1 injections (KO rhNRG1 ip; n=6 animalsper genotype; mean±SEM). FIG. 9D, Immunoblot of pooled tissue from atleast three different animals prepared from sciatic nerves, brachialnerves, trigeminal nerves and dorsal root ganglions (DRG).Nf1flox;Postn-Cre mutant animals received either vehicle controlinjections (KO vehicle ip) or rhNRG1 treatment (KO rhNRG1 ip) over 3months. Immunostaining for ErbB2, Akt, phospho-Akt (pAkt), c-Jun,Erk1/2, phospho-Erk1/2 (pErk1/2), myelin protein zero (MPZ), myelinbasic protein (MBP) and GAPDH as loading control (n=3).

EXAMPLES

The examples show that the myelination promoting function of NRG 1 as aninstructive signal to de-differentiated Schwann cells has beneficialeffects on schwannomas. In particular the examples show that byadministering the EGF domain of recombinant human Neuregulin1 (rhNRG1)the development of schwannoma and neurofibroma can be efficientlyprevented, reduced or reversed.

Materials and Methods Used in the Examples:

Experimental Animals

All animals had free access to food and water and were housed underconstant temperature and humidity conditions on a 12/12-h light/darkcycle. Nf2flox animals (RIKEN BioResource Centre) were used to obtaincombined conditional knockout of merlin in Schwann cells (PO-Cre line,The Jackson Laboratory, USA, stock 017928) and neurons (Nefh-Cre, TheJackson Laboratory, USA, stock 009102). Heterozygous knockout animals(PO-Cre;Nefh-Cre;Nf2^(fl/+)) were compared to wildtype littermates. Crerecombinase-specific genotyping was performed using the followingprimers: 5′-CCA CCA CCT CTC CAT TGC AC-3′ (forward) and 5′-ATG TTT AGCTGG CCC AAA TG-3′ (reverse) for PO-Cre (Feltri et al., 1999) as well as5′-GGG CCA CCG CGG ATA TAA AA-3′ (forward) and 5′-TGC GAA CCT CAT CACTCG TT-3′ (reverse) for Nefh-Cre recombinase (Hirasawa et al., 2001).Nf2flox;PO-Cre;Nefh-Cre animals were on a mixed C57BL/6-FVB/Nbackground.

Nf2flox;Postn-Cre mice were obtained by crossing Nf2flox animals withPostn-Cre transgene mice. The Postn-Cre transgene was detected by PCRanalysis with the following primers: P1 (CAT-TTG-GGC-CAG-CTA-AAC-AT) andP2 (CCC-GGC-AAA-ACA-GGT-AGT-TA). Nf2flox;Postn-Cre mice were on mixedFVB/NTac (Gehlhausen et al., 2015).

P0-Cre;Nefh-Cre;Nf2^(fl/+), Nf2flox;Postn-Cre or Nf1flox;Postn-Cremutant mice are herein also briefly referred to as ‘mutant’ mice orknockout (KO) mice

Sciatic Nerve Crush Injury

Unilateral injuries of sciatic nerves were accomplished according to apreviously described method (Bauder and Ferguson, 2012). In brief, 8 to10 week old mice were anesthetized using 2% isoflurane in 100% oxygen.Fur was then removed from one hind limb. After an appropriate incisionof the skin, the gluteal musculature was separated in order to revealthe right sciatic nerve. Using haemostatic forceps (Ultra FineHaemostat; #13021-12; tip width 0.6 mm; Fine Science Tools; Germany),the nerve was crushed once by the application of a defined pressure for20 s. The locking mechanism of the haemostatic forceps with a series ofinterlocking teeth ensured reproducibility and standardization of crushinjury. Finally, both the gluteal musculature and skin incision weresutured using appropriate surgical suture material.

rhNRG1 and Control Treatment

An EGF domain containing 7 kDa protein of recombinant human Neuregulin 1(“rhNRG1”) was purchased from Reprokine (#RKQ02297; USA). Protein wasfirstly resolved in ddH2O and further diluted in phosphate-bufferedsaline (PBS) to reach target concentrations. For systemicadministration, 10 μg rhNRG1 per kg body weight was intraperitoneallyinjected into mice every other day. For local administration, Spongostancollagen sponges (#2484887; Ethicon; Germany) were cut into cubes of 2mm edge length and incubated with 0.2 mg/mi rhNRG1 solution for one hourat room temperature prior to implantation. PBS solution without rhNRG1served as vehicle control.

Absorbable Collagen Sponge (ACS) Performance In Vitro.

Spongostan collagen sponges (#2484887; Ethicon; Germany) were cut intocubes of 2 mm edge length and incubated with a solution containing 0.1,0.5 or 1.0 mg/ml rhNRG1 for 1 hour. rhNRG1-soaked sponges weretransferred to a new dish of a 24-well plate filled with 0.5 ml PBSsolution every day. rhNRG1 concentration of each dish was asses usingstandard BCA protein assay. Sponges that were initially incubated withPBS only served as control.

Blood Sampling and Serum Separation

To measure peripheral exposure of rhNRG1 in mice, up to 50 μl blood wassampled from mice for four consecutive days via bilateral retro-orbitalplexus and facial veins. For serum preparation, collected blood wasallowed to clot at room temperature for 1 hour before spinning for 20minutes at 2,000 g.

Nrgl ELISA Kit

Blood serum levels of rhNRG1 before and after intraperitoneal injectionwere measured using a commercially available kit according tomanufacturer's instructions (#ab100614; Abcam; USA).

Single-Frame Motion Analysis (SFMA)

To evaluate locomotor function, mice were accustomed, in 3-4 trials, tobeam-walking 1 week prior to surgery. In this test, the animal walksvoluntarily from one end of a horizontal beam (length 1000 mm, width 40mm) towards its home cage located at the other end of the beam. For allmice, a rear view of one walking trial was captured once prior tosurgery and at different time-points after surgery with a Video cameraand stored on a personal computer in Audio Video Interleaved (AVI)format. The video sequences were examined using VirtualDub 1.6.19software. Selected frames, in which the animals were seen in definedphases of the step cycle, were used to measure the foot-base angle (FBA)as described in (Fey et al., 2010).

In Situ Tumour Size Quantification.

Three months after crush injury the right sciatic nerve of all animalswas exposed surgically in order to asses sciatic nerve diameter asindicator for tumour size. Documentation was performed by video-assistedmicroscopy for each animal. Selected frames from the video files (usingVirtualDub 1.6.19 software) were used to determine sciatic nervediameter using ImageJ.

Neuropathological Assessment and Evaluation

For histological workup, paraformaldehyd fixed nerve samples wereembedded in paraffin, cut at the site of the largest diameter andmounted as a tissue micro array. 4 μm thick cross sections were used forH&E staining and immunohistochemical labelling of 5100 (1:8000; Dako),PO (1:300; Bioss Antibodies), phospho-c-Jun (Ser73; 1:500; CellSignalling; 8752), ErbB2 (1:500; Cell Signaling) and Arginase-1 (1:200;Santa Cruz; H-52) in an automated Ventana stainer (Ventana MedicalSystems, USA) using standard antigen retrieval protocols (CC1st, nopretreatment for S100-protein). Onion-bulbs and schwannoma-likestructures were assessed in H&E stained slides.

Immunohistochemistry and Cell Quantification

Immunohistochemistry was performed as described in (Schulz et al.,2010). Briefly, paraffin-embedded, longitudinal sections of sciaticnerves were rehydrated, boiled in 10 mM sodium citrate buffer (pH9) for30 min in a microwave and subsequently treated with 0.5% Triton X-100for 10 min. Sections incubated in 0.2% gelatine and 2% goat serumdiluted in PBS at least 2 h. Sections submersed in the primary antibodysolution overnight at 4° C. The following primary antibodies were used:Myelin protein zero (P0; 1:200; Abcam; ab39375), neurofilament (1:200;BioLegend; SMI312), myelin basic protein (MBP; 1:500; Millipore;MAB384)), Ki-67 (1:200; eBioscience; Clone: SolA15), Iba-1 (1: 200;Wako), and p75 (1:200; Merck Millipore; AB 1554). After vigorouswashings, sections incubated with secondary antibody solution(Alexa488-, Alexa546- or Alexa647-conjugated anti-mouse, -rat, -chickenand -rabbit antibodies, 1:500 in PBS, Invitrogen) at room temperaturefor 2 h. Finally specimens washed in PBS, counterstained using DAPI (1μg/ml PBS, 10 min) dehydrated and embedded. DAPI-stained cell nuclei orIba positive cells were counted using ImageJ plugin ‘Particle Analysis’from images acquired with 10× magnification after standardizedbackground reduction and threshold setting.

Immunoblotting

Immunoblotting was performed as described in (Morrison et al., 2001).The following primary antibodies were used: phospho-c-Jun (Ser73;1:1000; Cell Signalling; 8752), anti-Nrgl (1:250; Santa Cruz; cloneC-20), anti-Erk (1:500; Cell Signalling), anti-phospho-Erk (T202/Y204;1:500; Cell Signalling), anti-ErbB2 (1:500; Cell Signalling), anti-GAPDH(1:1000; Santa Cruz; 6C5), anti-merlin (1:500; Santa Cruz; A19). Westernblot results were quantified using Gel analysis software by ImageJ.Density values were normalized to actin and appropriate controls oftransfection or wild-type tissue, respectively. In case ofphospho-specific detection of proteins, their acquired densities werereferred to signals derived from related pan-antibodies (e.g.phospho-Erk to Erk signals).

Morphometric Analysis of Nerve Sections

Analysis of axon calibre, myelination thickness and solidity factor wasconducted on semi-thin and ultra-thin sections of the sciatic nerveremoved from transcardially perfused mice. Mice were perfused with asolution containing 3% paraformaldehyde and 3% glutaraldehyde in 0.1 Mphosphate buffer (pH 7.4). Sections were postfixed for 1 h and kept infixative including 3% sucrose. Sections were obtained from the distalpart of the sciatic nerve. Sectioning and staining were performed asdescribed. Images of toluidine blue-stained semi-thin sections weretaken using an Axioskop 2 MOT (Carl Zeiss, Germany) equipped with a 100×immersion oil objective and an Olympus XC50 digital camera (Olympus,Germany). Standardized settings for camera sensitivity, resolution(2,576×1,932 pixels) and brightness of illumination were used for allmicrographs. Ultra-thin sections were analysed with an electronmicroscope (EM910, Carl Zeiss) equipped with an integrated TRS 1Kdigital camera (Carl Zeiss). Image analysis was conducted with ImageJversion 1.48u. RGB colour images obtained from semi-thin sections weresplit into single channels and the green channel was chosen formeasurements. The image was contrasted using the auto function. Axon andmyelin were circumscribed manually by the freehand selection tool. Basedon the measured areas, the thicknesses of the axons and myelin sheathswere calculated. All calculations and statistics were done in R(http://www.r-project.org/).

Quantification of Tumorlets in Nf2flox;Postn-Cre Mice and Tumors inNf1flox;Postn-Cre Mice.

PFA-fixed tissue blocks derived from rhNRG1 and control treated animalscontaining lower spinal cord, lumbar dorsal root ganglions (DRG) as wellas distal nerve proportions were cut in 4 planes per animal. Photographswere subsequently scanned and analyzed using the NanoZoomer DigitalPathology Software. The total area occupied by peripheral nerve tissueand dorsal root ganglion (DRG) tissue in mm² was quantified separately.Likewise, neoplastic tissue regions were identified and quantifiedseparately for peripheral nerves and DRG areas. All quantitativeanalyses of tumorlets in Nf2flox;Postn-Cre mice were performed inblinded manner.

Statistical Analysis

Comparisons between groups were made with unpaired t tests unless statedotherwise (SPSS software, Statistical Package for the Social Sciences,USA). For each experiment we calculated the p value (p), the t value (t)and the degree of freedom (df). Differences were considered significantwhen p<0.05. All values presented as means and their standard errors.

The following examples describe experimental results for rhNRG1treatment of WT and mutant mice, which show that systemic and localadministration of rhNRG1 effectively treats schwannoma and neurofibroma.Experimental procedures were performed as described in Example 1(materials and methods) and corresponding experimental data is depictedin FIG. 1-9 .

Example 1: rhNRG1 Treatment Animals does not Lead to Body WeightReduction, but Yields High Levels of Serum

All mice underwent the defined study protocol as shown in FIG. 1A. Crushinjury-induced schwannoma formation was provoked in 2-month-old mice.Subsequently, administration of rhNRG1 was performed both systemically(by intraperitoneal injection) and locally (by rhNRG1-containingabsorbable collagen sponges (ACS)). Vehicle saline solution was given tocontrol animals. In order to assess the bioavailability ofintraperitoneally injected rhNRG1 (10 μg per kg body weight), rhNRG1serum levels were evaluated. Strikingly, rhNRG1 levels reached a peak 2days after injection (FIG. 1B). Because rhNRG1 treatment at high doseswas reportedly accompanied by a decrease in body weight (Fledrich etal., 2014), we weekly checked the weight of systemically treatedanimals. A significant decline in body weight at the chosen dosage was,however, not observed (FIG. 1C).

Example 2: rhNRG1 Protein Treatment Improves Functional Recovery afterSciatic Nerve Crush

It was tested whether rhNRG1 protein replacement can improve functionalrecovery of mice over a time period of 8 weeks after sciatic nervecrush. Performing single-frame motion analysis (SFMA) showed thatsystemic rhNRG1 application to wildtype animals does not enhanceregeneration (FIG. 2A). However, Nf2flox;PO-Cre;Nefh-Cre mutant micereceiving continuous intraperitoneal rhNRG1 injections every other daydisplayed significantly improved recovery compared to mice withouttreatment or saline control (FIG. 2B).

In addition to the systemic administration of rhNRG1, the efficacy ofrhNRG1 on schwannoma growth was investigated when applied locally. Tothat end, biodegradable and biocompatible absorbable collagen sponges(ACS) were used to load either rhNRG1 protein or saline controlsolution. ACS have been successfully used for the locally-restricteddelivery of proteins in preclinical settings (Friess, 1998) and haveapplications in the clinical setting for hemostasis (Browder and Litwin,1986). Absorbable collagen sponges (ACS) cubes were incubated withsolutions containing different concentrations of rhNRG1 (FIG. 2D).Subsequent analysis concerning the amount of rhNRG1 protein secreted byACS over time in vitro showed a stable expulsion of rhNRG1 by thecollagen sponges (FIG. 2D). In vivo biostability analysis furtherdetermined that ACS cubes in close proximity to sciatic nerves werestill detectable two weeks after the implantation.

Consistently with the results for the systematic administration, thelocal administration of rhNRG1 using absorbable collagen sponges wasable to increase functional regeneration in Nf2flox;PO-Cre;Nefh-Cremutant mice compared to control treatment (FIG. 2C).

Example 3: rhNRG1 Protein Treatment Impedes the Development ofSchwannoma

Sciatic nerve crush injury was conducted on WT mice andNf2flox;PO-Cre;Nefh-Cre mutant mice as described above. Three monthsafter crush injury wildtype animals showed anatomically regular sciaticnerves with a mean diameter of about 1 mm (WT vehicle, FIG. 3A). Incontrast, the sciatic nerve of Nf2flox;PO-Cre;Nefh-Cre mutant micereceiving saline control injections appeared massively enlargedresulting in a 2-fold gain in nerve diameter to about 2 mm (KO vehicle,FIG. 3A). The systemic administration of rhNRG1, however, couldsignificantly ameliorate this nerve swelling and resulted in a reducednerve diameter of about 1.5 mm (KO rhNRG1, FIG. 3A). Slightly minoreffects were seen in Nf2flox;PO-Cre;Nefh-Cre mutant mice receiving alocal therapy of rhNRG1 using absorbable collagen sponges (rhNRG1 ACS)when compared to Nf2flox;PO-Cre;Nefh-Cre mutant mice receiving thecontrol therapy (FIG. 3B). 3 months after crush injury wildtype animalsshowed anatomically regular sciatic nerves with a mean diameter of about1 mm (WT vehicle ACS, FIG. 3B). The sciatic nerve ofNf2flox;PO-Cre;Nefh-Cre mutant mice receiving control Spongostancollagen sponges incubated with a PBS solution exhibited an enlargednerve diameter of about 1.8 mm after three months (KO vehicle ACS, FIG.3B). Local administration of rhNRG1 to Nf2flox;PO-Cre;Nefh-Cre mutantmice by treatment of Spongostan collagen sponges incubated with a 0.2mg/ml rhNRG1 solution reduced the nerve swelling and resulted in a nervediameter of about 1.4 mm after 3 month (KO rhNRG1 ACS, FIG. 3B).Moreover by determining the myelin thickness, structural integrity ofnon-crushed, intact nerves was assessed (FIG. 7 ). The long-termtreatment with rhNRG1 over 8 weeks had no obvious impact on themicroarchitecture of intact nerves as indicated by similar g-ratiovalues for rhNRG1 and PBS control treated animals.

Example 4: rhNRG1 Protein Treatment Leads to Differentiation ofDe-Differentiated Schwann Cells

Sciatic nerve crush injury was performed on WT andNf2flox;PO-Cre;Nefh-Cre mutant animals as described in Example 1. TheNf2flox;PO-Cre;Nefh-Cre mutant animals were intraperitoneally injectedevery day with 10 μg of rhNRG1 per kg body weight (KO rhNRG1). For acontrol a PBS solution was systemically administered to WT animals andNf2flox;PO-Cre;Nefh-Cre mutant animals (WT vehicle and KO vehicle).Sciatic nerve lysates were taken from animals after 3 month of treatmentupon which immunochemistry was performed as described in Example 1.

Neuropathological assessment of nerve tissue 3 months after crush injuryrevealed regular normal nerve composition in wildtype animals treatedwith saline control injections (FIG. 2A, left panel), accompanied bynormal expression of the Schwann cell differentiation markers S100 andmyelin protein zero (MPZ). Neuregulin1 and ErbB2 immunoreactivity waslocalized to axonal membrane and Schwann cells, respectively. The p75neurotrophin receptor (marker for immature non-myelinating Schwanncells) showed expression restricted to scattered locations only. Theputative schwannoma marker CPI-17, up-regulated in human schwannomas butno other PNS nerve tumors (Hagel et al., 2016), also showed only weakimmunostaining in sciatic nerves wildtype animals receiving controlinjections.

In contrast, nerves of vehicle-treated KO (PO-Cre;Nefh-Cre;Nf2^(fl/+))animals demonstrated large clusters of unordered Schwann cells withlarge concentric multilayered onion bulbs 3 months after nerve crushinjury (FIG. 2A, middle panel). S100 and MPZ expression appeared to beincreased in comparison to wildtype mice. Consistently, Neuregulin1 andErbB2 immunoreactivity was markedly enhanced in these nerves.Up-regulated p75 and CPI-17 expression suggests the presence of moreimmature non-myelinating Schwann cells and the classification asschwannoma, respectively.

Sciatic nerves of rhNRG1-treated KO (P0-Cre;Nefh-Cre;Nf2^(fl/+))animals, instead, showed almost regular regeneration 3 months aftercrush injury. S100 and MPZ immunoreactivity was restricted to Schwanncells with myelinating phenotype. Strikingly, the expression of bothNeuregulin1 and the receptor tyrosine kinase ErbB2 was greatly reducedfollowing systemic rhNRG1 treatment when compared with saline treatedanimals of the same genotype. Furthermore, the number of immatureSchwann cells appeared relevantly decreased due to rhNRG1 injections asindicated p75 staining. The immunoreactivity of the presumptiveschwannoma marker CPI-17 was also relevantly lower than in KO animalsreceiving control injections.

Sciatic nerve lysates taken from Nf2flox;PO-Cre;Nefh-Cre animals after 3months of rhNRG1 treatment demonstrated reduced expression of the myelinprotein zero (MPZ) as a marker for differentiated Schwann cells in bothknockout animal groups (KO vehicle and KO rhNRG1, FIG. 4B). Furthermore,the 17 kDa-myelin basic protein (MBP) isoform (Boggs 2006) showed aremarkable reduced expression in both knockout groups compared towildtype littermates (FIG. 4B). However, the 21.5-kDa MBP isoform, whichhas been repeatedly reported to be re-myelination-specific (Capello etal. 1997, He et al. 2012), showed a strong up-regulation in animalsreceiving treatment with rhNRG1 (KO rhNRG1). This result demonstrates anincreased Schwann cell differentiation upon rhNRG1 treatment.Consistently, c-Jun levels as marker for undifferentiated Schwann cellswas reduced in knockout animals receiving systemic rhNRG1 injections(FIG. 4B). ErbB2, one of the most abundantly expressed receptor tyrosinekinases in human schwannomas (Boin et al., 2014), shows high expressionin vehicle treated knockout animals but normal expression in rhNRG1treated mice (FIG. 4C). The macrophage number in the nerve tissue, asindicated by Iba-1 immunoblot (FIG. 4C), was not influenced by systemicrhNRG1 treatment.

Example 5: rhNRG1 Protein Treatment Reduces Tumor Growth ofPre-Established Schwannoma

Two cohorts of animals underwent crush injury-induced schwannomainduction at the right sciatic nerve as described above (FIG. 5A). Aftera period of three months without any treatment, the maximum sciaticnerve diameter was found to be equal in both groups (FIG. 5B). Thenknockout animals (Nf2flox;PO-Cre;Nefh-Cre mutant animals) receivedeither systemic rhNRG1 treatment or vehicle control applications forthree months (FIG. 5A). As a result of rhNRG1 treatment, tumor size wasmarkedly smaller compared to vehicle administration. (FIG. 5B)

Example 6: rhNRG1 Protein Treatment on Schwannoma Growth inNf2flox;Postn-Cre Mice

The in vivo results on the efficacy of rhNRG1 on schwannoma growth wastested as described in Example 1 however in a another animal model ofschwannoma disease, the well-established Nf2flox;Postn-Cre mouse line,wherein a conditional Nf2 gene deletion is facilitated by thePeriostin-Cre driver line (Nf2flox;Postn-Cre).

In this model all animals spontaneously develop spinal, peripheral andcranial nerve schwannomas over time (Gehlhausen et al., 2015).4-month-old mutant Nf2flox;Postn-Cre animals as well as theircorresponding wildtype littermates were either treated with salinecontrol injections or intraperitoneal rhNRG1 administration for threemonths (FIG. 8A). As a rhNRG1 dosage of 10 μg/kg was well tolerated inNf2flox;PO-Cre;Nefh-Cre animals, Nf2flox;Postn-Cre mice were treatedwith an increased dosage of 20 μg rhNRG1 per kg bodyweight. In order totest rhNRG1 efficacy, the phenotypic feature of Nf2flox;Postn-Cre mutantanimals to develop neoplastic Schwann cell proliferation areas(tumorlets) was deployed. Tumorlets are considered schwannoma precursorsin spinal and peripheral nerves (FIG. 8B). Quantification of the areaoccupied by tumorlets in relation to the total area of investigatednerve tissue revealed a significant reduction of tumorlet fraction inrhNRG1-treated animals as compared to control animals (FIG. 8C).

Example 7: rhNRG1 Protein Treatment Reduces Neurofibroma Growth inNf1flox;Postn-Cre Mice

Another closely related but not similar entity of Schwann cell tumorsare neurofibromas. As opposed to schwannomas, Schwann cells representthe primary neoplastic cell component of neurofibromas(Stemmer-Rachamimov et al., 2004; Rodriguez et al., 2012), but alsoincorporate a mixture of non-neoplastic components, including axons,perineurial cells, fibroblasts and inflammatory cells. In order to testa possible effect of rhNRG1 treatment on these tumors, which are atypical manifestation of the hereditary disease Neurofibromatosis type 1(NF1), 4-month-old mutant Nf1flox;Postn-Cre animals as well as wildtypelittermates were either treated with saline control injections orintraperitoneal rhNRG1 administration for three months (FIG. 9A).Analysis and quantification of neurofibromas originating from distalparts of the nervous system revealed a significant effect of rhNRG1treatment (FIGS. 9B and 9C). Biochemical analysis of nerve tissue fromNf1flox;Postn-Cre mice illustrate further a reduced ErbB2 expressionfollowing rhNRG1 treatment as well as enhanced Schwann celldifferentiation indicated by increased myelin protein zero (MPZ)expression (FIG. 9D).

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1. A method for treating and/or preventing a tumor of the nervous systemin a subject, comprising administering a nucleic acid molecule to thesubject, wherein the nucleic acid molecule encodes a polypeptide thatcomprises an EGF-like domain of a neuregulin protein.
 2. The methodaccording to claim 1, wherein the polypeptide is a soluble fragment of aneuregulin protein.
 3. The method according to claim 1, wherein thepolypeptide comprises an amino acid sequence according to one of SEQ IDNO 9-14 or an amino acid sequence with an identity of at least 80% toany one of SEQ ID NO 9-14.
 4. The method according to claim 1, whereinthe polypeptide consists of an amino acid sequence according to SEQ IDNO 9 or of an amino acid sequence with an identity of at least 80% toSEQ ID NO
 9. 5. The method according to claim 1, wherein the tumor is atumor of the cranial or peripheral nerves.
 6. The method according toclaim 1, wherein the tumor is a malignant nerve sheath tumor.
 7. Themethod according to claim 1, wherein the tumor is a schwannoma.
 8. Themethod according to claim 1, wherein the tumor is a neurofibroma.
 9. Themethod according to claim 1, for treating and/or preventing medicalconditions associated with one or more of genetic deficiency inneurofibromatosis type 1, neurofibromatosis type 2, or neurofibromatosistype
 3. 10. The method according to claim 1, wherein the nucleic acidmolecule comprises a gene therapy vector.
 11. The method according toclaim 10, wherein the gene therapy vector is a viral vector.
 12. Themethod according to claim 11, wherein viral vector is selected from thegroup consisting of a moloney murine leukemia virus (MMLV), anadenoviral vector, a lentiviral vector, an adenovirus-associated viral(AAV) vector, a pox virus vector, a herpes simplex virus vector or humanimmunodeficiency virus vector (HIV-1).
 13. The method according to claim10, wherein the gene therapy vector is a non-viral vector.
 14. Themethod according to claim 13, wherein the gene therapy vector istransferred by electroporation or injection of nucleic acid moleculeinto the subject.
 15. The method according to claim 13, wherein the genetherapy vector is a plasmid DNA expression vector driven by a eukaryoticpromoter, or liposomes encapsulating the vector.
 16. The methodaccording to claim 10, wherein the gene therapy vector is configured forinducible or conditional expression of the polypeptide.
 17. The methodaccording to claim 1, comprising administering a cell to the subject,wherein the cell is genetically modified to comprise the nucleic acidmolecule according to claim
 1. 18. The method according to claim 1,wherein the nucleic acid molecule is configured to incorporate into thegenome of a host cell of the subject.
 19. The method according to claim1, wherein the composition is administered intravenously to the subject.20. The method according to claim 1, wherein the composition isadministered locally to the site of the tumor.